Variation in transplacental transfer of tyrosine kinase inhibitors in the human perfused cotyledon model.

BACKGROUND: The use of tyrosine kinase inhibitors (TKis) during pregnancy in humans remains rare, and little data are available on their transplacental passage. Erlotinib and gefitinib are the first-line targeted therapy in case of stage IV nonsmall-cell lung cancer with an EGFR-activating mutation. There are no data available regarding the comparative use of these TKis in pregnant patients. We aimed to compare the transplacental transfer of gefitinib, imatinib and erlotinib, using the ex vivo method of human perfused cotyledon, and to determine the placental accumulation of TKis. MATERIALS AND METHODS: Term placentas were perfused after delivery with gefitinib, imatinib and erlotinib at targeted maternal concentrations around the steady-state plasma trough concentration (i.e. 500, 1000 and 1500 ng/ml, respectively). Samples from fetal and maternal circulations were collected in order to monitor TKis concentrations. Main transfer parameters such as fetal transfer rate (FTR), clearance index (CI) and placental uptake were assessed. RESULTS: Mean FTR of gefitinib, imatinib and erlotinib were 16.8%, 10.6% and 31.4%, respectively. Mean CI of gefitinib, imatinib and erlotinib were 0.59, 0.48 and 0.93, respectively. Placental uptake in cotyledon was 0.030% %, 0.010% and 0.003% for gefitinib, imatinib and erlotinib, respectively, corresponding to a mean mass of 27.7 µg for gefitinib, 15.7 µg for imatinib and 6.8 µg for erlotinib. CONCLUSION: The results suggest that TKis cross the placenta at therapeutic level. Particularly, erlotinib crosses the placenta at a higher rate than gefitinib or imatinib. All of them have a very low placental uptake. These data may suggest that gefitinib should be preferred to erlotinib for the treatment of pregnant woman with lung cancer harboring an EGFR-activating mutation, during the second and third trimesters of pregnancy.

Candidate epitopes for measurement of hCG and related molecules: the second ISOBM TD-7 workshop.

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, hCGß, hCGßn, hCGßcf, 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 hCGß-, 8 hCGα-, and 13 αß-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (<0.1%; epitope ß1), 12 with low (<1.0%; epitopes ß2/4), and 13 with high (>>1%; epitopes ß3/5) hLH cross-reactivity. The majority of hCGß epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of ß-sheet loops 1 and 3 (epitopes ß2-6; 27 mAbs) and the second around the cystine knot (e.g., epitopes ß1, ß7, and ß10; 9 mAbs). Four mAbs recognized epitopes on hCGßcf-only (e.g., epitopes ß11 and ß13) and six mAbs epitopes on the remote hCGß-carboxyl-terminal peptide (epitopes ß8 and ß9 corresponding to amino acids 135-144 and 111-116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGß-only, or hCG together with hCGß or hCG together with hCGß and hCGßcf. Sandwich assays that measure hCG plus hCGß and eventually hCGßcf 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 hCGßcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope ß1 (Asp10, Asp60, and Gln89) in combination with epitopes ß2 or ß4 located at the top of ß-sheet loops 1 + 3 of hCGß involving aa hCGß20-25 + 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.

Influence of the multidrug transporter P-glycoprotein on the intracellular pharmacokinetics of vandetanib.

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.

Iodide transporters expression in early human invasive trophoblast.

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.

Somatic RAS mutations occur in a large proportion of sporadic RET-negative medullary thyroid carcinomas and extend to a previously unidentified exon.

CONTEXT: 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. OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Inhibition of P-glycoprotein functionality by vandetanib may reverse cancer cell resistance to doxorubicin.

P-glycoprotein belongs to the ATP binding cassette transporters, responsible for the multidrug resistance of cancer cells. These transporters efflux hydrophobic drugs outside cells and decrease their therapeutic efficacy. The aim of this study was to investigate the effect of vandetanib, an oral tyrosine kinase inhibitor of EGFR, VEGFR 2 and RET kinases, on the functionality of P-gp after a 24h-treatment at therapeutic concentration (2µM), and its ability to increase the cytotoxicity of chemotherapeutic agents in multidrug resistance cancer cells. In this study we found that IGROV1-DXR and IGROV1-CDDP cells were resistant to doxorubicin and cisplatin respectively, compare to parental cell line IGROV1. The parental sensitive and the two resistant cell lines similarly expressed MRP1 and did not express BCRP. Moreover, in contrast to the IGROV1 and IGROV1-CDDP cells, IGROV1-DXR cell line overexpressed P-gp. Functional activity studies demonstrated that MRP1 was not functional and the MDR phenotype in IGROV1-DXR cells was linked to P-gp functionality. Results also showed that vandetanib reversed resistance to doxorubicin in IGROV1-DXR cells, but not to cisplatin in IGROV1-CDDP cells. After 24h of treatment, vandetanib increased the accumulation of rhodamine 123 and calcein AM, demonstrating a functional inhibition of the transporter. In IGROV1-DXR cell line, vandetanib reverse resistance to doxorubicin by inhibiting the functionality of P-gp. In conclusion, vandetanib should be an option for drug combination in patients already developing a P-gp mediated multidrug resistance.

ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence.

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, HP1ß 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.

Do histological, immunohistochemical, and metabolic (radioiodine and fluorodeoxyglucose uptakes) patterns of metastatic thyroid cancer correlate with patient outcome?

The aim of this study is to search for relationships between histology, radioiodine ((131)I) uptake, fluorodeoxyglucose (FDG) uptake, and disease outcome in patients with metastatic thyroid cancer. Eighty patients with metastatic thyroid cancer (34 males, 46 females, mean age at the time of the diagnosis of metastases: 55 years) were retrospectively studied. All patients were treated with radioactive iodine and evaluated by FDG-positron emission tomography (PET). Primary tumor tissue sample was available in all cases. Forty-five patients (56%) had a papillary, 12 (15%) a follicular, and 23 (29%) a poorly differentiated thyroid cancer. Cellular atypias, necrosis, mitoses, thyroid capsule infiltration, and vascular invasion were frequently detected (70, 44, 52, 60, and 71% respectively). Metastases disclosed FDG uptake in 58 patients (72%) and (131)I uptake in 37 patients (45%). FDG uptake was the only significant prognostic factor for survival (P=0.02). The maximum standardized uptake value and the number of FDG avid lesions were also related to prognosis (P=0.03 and 0.009). Age at the time of the diagnosis of metastases (P=0.001) and the presence of necrosis (P=0.002) were independent predictive factors of FDG uptake. Radioiodine uptake was prognostic for stable disease (P=0.001) and necrosis for progressive disease at 1 year (P=0.001). Histological subtype was not correlated with in vivo tumor metabolism and prognosis. In conclusion, FDG uptake in metastatic thyroid cancer is highly prognostic for survival. Histological subtype alone does not correlate with (131)I/FDG uptake pattern and patient outcome. Well-differentiated thyroid cancer presenting histological features such as necrosis and FDG uptake on PET scan should be considered aggressive differentiated cancers.

Gene expression profiling identifies Fibronectin 1 and CXCL9 as candidate biomarkers for breast cancer screening.

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 microg ml(-1) for patients and 125 microg ml(-1) for volunteers (P<0.001). Areas under the curve for BC diagnosis were 0.78 and 0.62 for Fibronectin 1 and CXCL9 respectively. A combined score including Fibronectin 1 and CXCL9 dosages presented 53% of sensitivity and 98% of specificity. Similar performances were observed for ER-negative tumours. CONCLUSIONS: This study suggests that Fibronectin 1/CXCL9 dosage in serum could screen a significant rate of BC, including ER-negative, and that differential gene expression analysis is a good approach to select candidate biomarkers to set up blood assays cancer screening.

[KRAS status versus EGFR status in lung cancer therapy]. / Détermination du statut KRAS associé au statut EGFR pour la prise en charge thérapeutique des cancers bronchiques.

Interest for development of molecular biomarkers tends to increase in clinical management of lung cancer. Indeed implementation in clinics of new molecules targeting epithelial growth factor (EGFR) such as erlotinib or gefitinib, rise several questions regarding the potential value of EGFR and KRAS mutations to predict therapeutic response. In the current review, we discuss the utilization of such biomarkers regarding published clinical data and the possibilities versus limitations associated with analyses performed in molecular laboratory on a daily setting basis.

[Evaluation of an immunoassay for calcitonin]. / Evaluation d'une methode de dosage de la calcitonine par chimio luminescence dans le suivi des cancers medullaires de la thyroïde.

INTRODUCTION: The calcitonin is the most specific and the most sensitive marker of medullary thyroid carcinoma (MTC) both for screening and postoperative follow-up of the patients. Its measurement is made either remotely , or after stimulation of pentagastrin secretion which the answer is amplified at the carrier of CMT. The aim of this study was to estimate a chimioluminescent method by comparing it with an immunoradiological method, manual, used as reference. Correlation study was done. MATERIALS AND METHODS: Two hundred and sixty three serums (263) were tested among which 64 resulting of healthy subjects and 199 resulting of patients affected) by medullary thyroid carcinoma. Statistical analysis of results was made by a study of correlation with the software OriginLab version 7.0. The manual technique used as reference method is radioimmunological (Elsa hCT, international Cisbio, Gif on Yvette, France). It was compared with a chimioluminescent technique (Nichols Advantage, Nichols Institute Diagnostics, CA, the USA). RESULTS: The coefficients of correlation obtained between both tests were: r = 0.76 (exactness study), r = 0.91 (after stimulation), r = 0.95 and 0.79 (staged samples), r = 0.99 (M TC patients). CONCLUSIONS: Both techniques correlate strictly and significantly. The correlation coefficients we obtained show us that Nichols Advantage Calcitonin is completely reliable and sensitive for the measurement of the hCT in the follow-up of the CMT.

[Comparison of three chemiluminescent methods with a radioimmunoassay for thyroglobulin measurement in the follow-up of differentiated thyroid cancer]. / Comparaison de trois methodes chimioluminescentes avec une methode radioimmunologique de dosage de la thyroglobuline dans le suivi des cancers differencies de la thyroide.

INTRODUCTION: Serum thyroglobulin measurements play an integral role in clinical evaluation of patients with thyroid cancer. Serum thyroglobulin is a highly specific and sensitive tumor marker for detecting persistent or recurrent thyroid cancer but also for monitoring clinical status. Actually, chemiluminescent methods gain ground on the radioimmunological methods because they offer the practical advantage of a shorter incubation time, a wider range of measured values and a reagent marked antibody more stable, less fragile than those used on radioimmunoassay. The aim of this study was to compare, by correlation study, three chemiluminescent methods to the reference radioimmunological method usually used in laboratories. MATERIALS AND METHODS: Thyroglobulin was measured in 203 patients by the 3 following analyzers: Nichols Advantage (Nichols Institute Diagnostics, CA, USA), Immulite 2000 ( DPC Roche, Siemens, Los Angeles, USA) and Elecsys 2010 (Roche Diagnostics, Manheim, Germany); and by manual method (SELco Tg (Medipan Diagnostica, Berlin, Germany). Correlation analysis with OriginLab software version 7.0 was performed in order to compare thyroglobulin distribution values measured by the different methods. RESULTS: Correlation coefficients obtained were for Medipan/ Immulite 2000: 0.95 (n = 80); for Medipan/Elecsys: 0.97 (n = 31); for Medipan/Advantage: r = 0.96 (n = 73). CONCLUSIONS: Chemioluminescent technics we studied could be validly used in patients without anti-thyroglobulin antibody. The correlation coefficients we obtained allow us to select one of these automated methods after their performance was studied.

Expression of early placenta insulin-like growth factor in breast cancer cells provides an autocrine loop that predominantly enhances invasiveness and motility.

Early placenta insulin-like growth factor (EPIL) is expressed by a subpopulation of the Her2-positive SKBR3 breast cancer cell line displaying high motility and transendothelial invasiveness in vitro, as recently shown by our group. As a consequence of this, we established cellular models by generating an EPIL-overexpressing SKBR3 cell line, knocked down EPIL by adding specific small interfering RNA (siRNA) to those cells and produced EPIL-enriched and depleted serum-free culture media. EPIL-expressing cells as well as EPIL-induced SKBR3 cells acquired a high capacity for transendothelial invasiveness. We observed a thin and outspread morphology caused by enhanced formation of lamellipodia, i.e. protrusions in the initial phase of motility. In parallel, Her2-positive MDAHer2 breast cancer cells also showed increased invasiveness when induced by EPIL-conditioned medium. A downstream signaling impact of EPIL could be observed in the form of reduced phosphorylation of Her2, erk1/2 and akt, while phospholipase Cgamma1 phophorylation remained unaffected. As an in vivo model for highly motile tumor cells, Paget's disease of the nipple showed simultaneous EPIL and Her2 expression upon immunohistochemical examination using specific antibodies. Such experimental data have been translated to a clinical setting by using a prognostic tissue microarray established from 603 breast cancer cases. Survival data analysis found a significant association between expression levels of EPIL and 5-year overall survival that was dose dependent: EPIL (negative) 84%, EPIL (moderately positive) 77%, EPIL (strongly positive) 48% (P < 0.005). One particular subgroup (7.6% of the cases with full clinical records) that comprised tumors simultaneously expressing EPIL and Her2 represented patients with the poorest 5-year overall survival. The results suggested that EPIL might be a cancer cell-produced growth factor that influences lateral Her2 signaling. Moreover, EPIL may be induced by factors apart from Her2 and may independently provide signaling for cancer invasion and motility.

Differential expression of galectin 3 in solid cell nests and C cells of human thyroid.

AIMS: To provide new insights into characterising solid cell nests and gain information that might help distinguish between solid cell nests and C cells. METHODS: Thyroid tissue specimens from patients who had undergone prophylactic thyroidectomy for familial medullary thyroid cancer were immunostained for calcitonin, carcinoembryonic antigen, and galectin 3. RESULTS: Solid cell nests displayed a strong and diffuse staining for carcinoembryonic antigen and galectin 3, but not for calcitonin. C cells located at the periphery of solid cell nests and in neighbouring follicles expressed both calcitonin and carcinoembryonic antigen but not galectin 3. These three markers were positive in medullary thyroid carcinoma. CONCLUSION: Galectin 3 immunoreactivity permits a better characterisation and differentiation between solid cell nests and C cells, avoiding the misidentification of two biologically and clinically different thyroid structures.

The ISOBM TD-7 Workshop on hCG and related molecules. Towards user-oriented standardization of pregnancy and tumor diagnosis: assignment of epitopes to the three-dimensional structure of diagnostically and commercially relevant monoclonal antibodies directed against human chorionic gonadotropin and derivatives.

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 chorionic gonadotropin (hCG) and its derivatives, and to consider how this information could be used to improve comparability of immunoassay results for these analytes. In this multicenter study, 27 MAbs have been characterized in detail as to their main and fine specificities by direct binding-, competitive- and sandwich-RIA, -ELISA, BIAcore and Western blotting. Antigens used in the study included the upcoming first WHO reference reagents for immunoassay, i.e. nick-free hCG (hCG), nicked hCG (hCGn), hCG alpha-subunit (hCGalpha), hCG beta-subunit (hCGbeta), nicked hCG beta-subunit (hCGbetan), hCG beta-core fragment (hCGbetacf), synthetic peptides of hCGbeta C-terminal peptide (hCGbetaCTP), and homologous hormones, luteinizing hormone (LH) and subunits (LHbeta) from various species. Correct classification of blinded internal controls demonstrated the reliability of the MAb referencing approach. Three-dimensional molecular epitope assignment was possible in many instances by comparing immunoreactivity of the ISOBM MAbs (n = 27) to a large panel of MAbs (n = 18) previously well characterized in the Innsbruck (P.B.) and Paris (J.M.B.) laboratories. All three major antibody specificities (alpha, n = 1; beta, n = 21; alphabeta, n = 5) were represented in the TD-7 MAb panel. HCGbeta MAbs could further be subdivided into (i) those recognizing hCGbeta only (epitopes: beta(6), n = 1; beta(7), n = 2; beta(14), n = 1) and (ii) those recognizing hCGbeta + hCG (beta1, beta2, beta4, beta5, n = 10; beta8 and beta9, n = 9). Members of the latter group were specific either for hCG + hCGbeta + hCGbetacf (beta1, n = 3) or hCG + hCGbeta + hCGbetaCTP (beta8, n = 6; beta9, n = 1) or in addition to hCG + hCGbeta + hCGbetacf recognized hLH/hLHbeta to a minor (beta2, n = 3; beta4, n = 3) or similar degree (beta5, n = 1). Epitopes were (i) located on the first and third loops protruding from the cystine knot of hCGbeta (beta2-beta6, aa hCGbeta20-25 and 68-77), (ii) presumably centered around the knot itself (beta1), or (iii) on hCGbetaCTP (epitope beta8 = hCGbeta141-144, beta9 = hCGbeta113-116). The ISOBM panel of MAbs represents all major epitope specificities suitable for the design of specific sandwich immunoassays. High analyte variability in serum and urine during the course of pregnancy and tumor development favors certain epitope combinations. For routine diagnostic purposes, assays recognizing a broad spectrum of hCG/hCGbeta variants such as hCG + hCGn + hCGbeta + hCGbetan + hCGbetacf + -CTPhCG + -CTPhCGbeta may be useful. Low cross-reactivity against related glycoprotein hormones (e.g. hLH) and their derivatives is mandatory. These criteria are best met by combinations of MAbs directed against epitopes located around the cystine knot (beta1) and against those encompassing the top of loops 1 and 3 on hCGbeta (beta2, beta4). The first WHO reference reagents for immunoassay of hCG and hCG-related molecules being prepared by the IFCC should facilitate characterization of what assays for 'hCG' are measuring. The next step towards improving between-laboratory comparability of measurements of hCG/hCG derivatives in pregnancy and oncology is provided by results of this TD-7 Workshop.

Medullary thyroid carcinoma as part of a multiple endocrine neoplasia type 2B syndrome: influence of the stage on the clinical course.

BACKGROUND: Multiple endocrine neoplasia type 2B (MEN 2B) is an exceptional syndrome, for which the optimal age of thyroidectomy is poorly established and the course of medullary thyroid carcinoma (MTC) is ill-defined. PATIENTS: All the 18 patients with a MEN 2B syndrome examined at the Institut Gustave Roussy were included in a single-center retrospective study. RESULTS: There were 9 men and 9 women with a mean age of 13 years (range, 2-27 years) at diagnosis. The diagnosis of MTC was based on the presence of a thyroid nodule or involved neck lymph nodes and on dysmorphic features of MEN 2B in 60% and 40% of the cases, respectively. The classic M918T mutation in exon 16 was found in the 16 patients in whom it was investigated. At diagnosis, 2 patients had Stage I MTC, 15 patients had Stage III, and 1 patient had Stage IV disease. T1 MTC was found in 4 patients aged 2.1-3.7 years. However, two of these patients already had N1 disease. One patient with Stage I MTC, aged 3.4 years and 2 patients with Stage III disease, aged 14 and 25 years, had undetectable basal calcitonin (CT) after initial surgery. During follow-up, basal CT became detectable in one of three patients. Among the 15 other patients with an elevated postoperative CT level, metastases were demonstrated in 5 patients after a mean follow-up of 2 years. Five patients died, three of MTC, one of the MEN 2B syndrome, and one of intercurrent disease. Five- and 10-year overall survival rates were 85% and 75%, respectively. CONCLUSIONS: This study confirms the need for early treatment of MTC in patients with the MEN 2B syndrome, preferably within the first 6 months of life. The phenotype of MTC occurring in the MEN 2B syndrome was not more aggressive than sporadic MTC or MTC occurring in other familial syndromes.

Sodium/iodide symporter (NIS) and pendrin are expressed differently in hot and cold nodules of thyroid toxic multinodular goiter.

OBJECTIVE: The expression of two iodide transporters, the sodium/iodide symporter (NIS) and pendrin, was analyzed in thyroid tissues of patients with toxic multinodular goiter (TMNG) and non-toxic multinodular goiter (MNG). METHODS: The levels of NIS and pendrin proteins were analyzed in total protein extracts from nodular and non-nodular tissues by Western blot. RESULTS: In tissue samples from TMNG, we found an increased expression of NIS (2.5-fold) in the hot nodules, and similar levels between cold nodules and non-nodular tissues. In contrast, the levels of pendrin were slightly increased in both hot and cold nodules from TMNG, and decreased (about twofold) in cold nodules from MNG. We also noticed that there was no relationship between NIS and pendrin expression. CONCLUSIONS: Our data demonstrate that hot nodules from TMNG express a higher number of iodide transporters (mainly NIS), whereas cold nodules from TMNG, but not from MNG, show levels of the two proteins comparable with normal tissue, suggesting a role in vivo of TSH in maintaining the expression of NIS and pendrin protein in normal thyroid tissue. Finally, different mechanisms are involved in the regulation of NIS and pendrin expression.

[Standards, options and recommendations for blood tumor markers in thyroid cancers]. / Standards, Options et Recommandations: marqueurs tumoraux seriques des cancers de la thyroide.

CONTEXT: The "Standards, Options and Recommendations" (SOR) project, started in 1993, is a collaboration between the Federation of the French Cancer Centers (FNCLCC), the 20 French Cancer Centers and specialists from French Public Universities, General Hospitals and Private Clinics. The main objective is the development of clinical practice guidelines to improve the quality of health care and outcome for cancer patients. The methodology is based on literature review and critical appraisal by a multidisciplinary group of experts, with feedback from specialists in cancer care delivery. OBJECTIVES: To define, according to the definitions of the Standards, Options and Recommendations project, the characteristics of the main tumor markers in thyroid cancer and the potential role of these markers in the management of patients with this malignancy. METHODS: Data were identified by searching Medline and the personal reference lists of members of the expert groups. Once the guidelines were defined, the document was submitted for review to 55 independent reviewers, and to the medical committees of the 20 French Cancer Centers. RESULTS: The main recommendations are: 1) Thyroglobulin is a serum tumor marker for the monitoring of operated thyroid differentiated neoplasms (standard). 2) It is essential to know if the patient is under TSH stimulation or under thyroid suppression therapy to interpret thyroglobulin results (standard). 3) Thyroglobulin assay must be performed regularly during the monitoring of differentiated thyroid neoplasms (standard, level of evidence B2), should be coupled with the measurement of anti-thyroglobulin antibodies concentration using a sensitive method (standard, level of evidence B2). 4) Thyroglobulin assay should not be performed to detect or diagnose differentiated thyroid neoplasms (standard, level of evidence B2). 5) The methods used to assay thyroglobulin must have a limit of detection lower than 3 mug.l- 1 (standard, expert agreement). 6) Calcitonin is a marker for medullary thyroid cancer (standard). 7) Its assay, associated with RET gene study if indicated, enables medullary thyroid cancer to be diagnosed. 8) The pentagastrin test is essential to diagnose familial forms of medullary thyroid cancer. 9) All analyses for each patient must be performed in the same laboratory, using the same technique (standard, expert agreement). 10) Calcitonin and carcinoembryonic-antigen are serum markers for the monitoring of medullary thyroid cancer and allow the detection of recurrent disease (standard).