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Background Patients with high-risk gestational trophoblastic neoplasia (GTN) need multi-agent chemotherapy to be cured. The most common regimen is etoposide (E), methotrexate (M) and actinomycin D (A), alternating weekly with cyclophosphamide (C) plus vincristine (O) (EMA/CO). Cisplatin (P) is a very active drug, but it is usually restricted to second-line therapies. Herein, we report the results of a cisplatin-based therapy: APE (actinomycin D, cisplatin, and etoposide). Patients and methods The efficacy and safety of APE for high-risk GTN (defined by Institut Gustave-Roussy (IGR) criteria and/or an International Federation of Gynaecology and Obstetrics (FIGO) score >6) are reported. Patients with brain metastasis or placental-site trophoblastic tumour were excluded. Results Between 1985 and 2013, 95 patients were treated with APE for high-risk GTN: 59 patients as first-line, 36 as ⩾2nd-line therapy. There was 94.7% complete remission, though five patients relapsed. One patient died from GTN after multiple lines of chemotherapy. The five-year overall survival rate (median follow-up 5.7 years) was 97% (95% confidence interval (CI): 91–99%). No death from toxicity occurred. Long-term, six grade-1 neuro-toxicities, three grade-1 and two grade-2 oto-toxicities, and one grade-1 renal toxicity were recorded. One patient developed AML-M4 after APE and EMA/CO. Thirty-four of 35 women, who wished to become pregnant, succeeded and all had at least one live birth. Conclusion With a 97% long-term overall survival rate, limited long-term toxicity, and an excellent reproductive outcome, APE could be regarded as an alternative option to EMA/CO as a standard therapy for high-risk GTN.

CA15-3, a peptide derived from MUC-1, an hormonally-regulated protein, is the most widely used serum marker of breast cancer. CA15-3 level increases at the metastatic phase in 50-80% breast cancer patients. Although rise of CA15-3 precede symptoms of metastasis by a mean time of 2-9 months, current international guidelines do not recommend its routine use for screening for metastases because of moderate sensitivity and absence of clinical impact. We conducted a retrospective study among all patients with metastatic breast cancer seen by three senior breast oncologists during a 4-month period. We evaluated correlation of CA15-3 level at the time of metastatic relapse with ER, PgR and Her2 expressions, tumor type, size and nodal status at initial diagnosis, and sites of metastases. CA15-3 was increased in 168/272 patients (62%) at diagnosis of metastases. ER/PgR positivity was strongly correlated with elevated CA15-3 at this time (P < 0.0001). CA 15-3 was elevated in 69% of the cases of HR+ Her2- primary tumors at time of metastatic relapse. It was elevated in 56% of HR+ Her2+++, 46% of HR- Her2+++ cases and only in 41% of triple-negative cases (P = 0.003). these data confirm that CA 15-3 is very variably elevated at time of metastatic relapse of breast cancer, and this is dependant on HR status.

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

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

Background: Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations. Methods: Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA). Results: The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 ± 16 U/ml vs. 700 ± 63 U/ml, P

Alternative splicing of the primary transcript of the CALC I gene in thyroid C-cells results predominantly in calcitonin (CT) mRNA (exons 1-4), whereas CGRP mRNA (exons 1, 2, 3, 5, and 6) is mainly produced in neuronal cells. The CT mRNA encodes for a protein precursor containing an amino terminal peptide, CT, and a carboxyl terminal peptide (CCP I). CGRP precursor is composed of the same amino terminal peptide and CGRP. Recently we reported the presence of a third mature transcript of the CALC I gene in human medullary thyroid carcinoma (MTC) tissues. This transcript encodes for a precursor containing the amino terminal peptide CT and a novel carboxyl terminal peptide, CCP II. This finding was further confirmed in the TT-cell line derived from a human MTC. We produced monoclonal antibodies against CCP II and developed a rapid and specific immunofluorescence method for this peptide. We demonstrated CCP II-specific immunoreactivity in TT-cells and in MTC tissues. CCP II labeling was relatively homogeneous in contrast to CT and CGRP, which presented striking heterogeneity for intensity of labeling. Therefore, CCP II mRNA is translated in tumor cells in an apparently constitutive way.

From medium conditioned by 3T3 cells, we had previously purified an inhibitory factor of Mr 45 kDa which we termed IDF45 (inhibitory diffusible factor). The protein was able to 100% inhibit stimulation induced in CEF by 1% calf serum and to reversibly prevent cell growth. We then demonstrated that IDF45 was an IGF-binding protein. Our results suggested that IDF45 was a bifunctional molecule able to bind IGF and to inhibit DNA synthesis stimulated by this hormone, but also to inhibit stimulation of DNA synthesis induced by another growth factor in serum. Indeed, its N terminal amino acid sequence has great homology with that of IGFBP-3 and IDF45 is now proposed to be named IGFBP-3 (mouse IGF binding protein). Present results show that Ha-ras transfected 3T3 cells (EJ cells), like 3T3 cells, secrete a mIGFBP-3 molecule. In addition, transfected cells secrete a doublet of an IGF-binding protein (IGFBP-28) of Mr 28 kDa which is not secreted by untransformed 3T3 cells. IGFBP-28 has been purified and characterized in this work. Various results suggest that IGFBP-28 is not a degradation product of mIGFBP-3. Its N terminal amino acid sequence was different from that of mIGFBP-3. IGFBP-28 inhibited DNA synthesis stimulated by IGF-I, but much more IGFBP-28 protein than mIGFBP-3 was required to prevent this stimulation. In agreement with this result, IGFBP-28 has low affinity for IGF-I. In contrast, IGFBP-28 has high affinity for IGF-II. Like mIGFBP-3, IGFBP-28 was able to inhibit the stimulation induced by serum in CEF and to reversibly prevent growth, though with a specific activity lower than that of mIGFBP-3. It has also the capacity to inhibit stimulation of DNA synthesis induced by high molecular weight serum proteins depleted in IGF-I and II. In conclusion we have shown that transformation of 3T3 cells with Ha-ras induced the synthesis of a new IGF binding protein in medium conditioned by normal 3T3 cells. Our results suggest that IGFBP-28 like mIGFBP-3 is a bifunctional protein able to inhibit stimulation induced by IGF and by serum proteins different from IGFs.

CA15-3, a peptide derived from MUC-1, an hormonally-regulated protein, is the most widely used serum marker of breast cancer. CA15-3 level increases at the metastatic phase in 50-80% breast cancer patients. Although rise of CA15-3 precede symptoms of metastasis by a mean time of 2-9 months, current international guidelines do not recommend its routine use for screening for metastases because of moderate sensitivity and absence of clinical impact. We conducted a retrospective study among all patients with metastatic breast cancer seen by three senior breast oncologists during a 4-month period. We evaluated correlation of CA15-3 level at the time of metastatic relapse with ER, PgR and Her2 expressions, tumor type, size and nodal status at initial diagnosis, and sites of metastases. CA15-3 was increased in 168/272 patients (62%) at diagnosis of metastases. ER/PgR positivity was strongly correlated with elevated CA15-3 at this time (P0.0001). CA 15-3 was elevated in 69% of the cases of HR+ Her2- primary tumors at time of metastatic relapse. It was elevated in 56% of HR+ Her2+++, 46% of HR- Her2+++ cases and only in 41% of triple-negative cases (P = 0.003). these data confirm that CA 15-3 is very variably elevated at time of metastatic relapse of breast cancer, and this is dependant on HR status.

A case of maternal generalized edema with hyponatremia, hypoosmolality and secondary hyperaldosteronism was associated with pseudomolar plasma human chorionic gonadotropin (hCG) concentrations in a case of fetal and placental hydrops due to parvovirus B19 infection. Digoxigenin in situ hybridization techniques were effective in demonstrating parvovirus B19 infection on fixed tissues. Hydropic changes in the placenta may have massively increased the maternal plasma hCG concentration with subsequent fluid imbalance leading to maternal hydrops mimicking molar pregnancy.

Hyperacute rejection (HAR) of discordant xenografts in the pig-to-human combination can be prevented using tranplants expressing transgenic molecules that inhibit human complement. Hypodermin A (HA), a serine esterase that degrades C3, was tested in the guinea-pig-to-rat and in the pig-to-human combinations.Hypodermin A was tested in vitro, ex vivo, and in vivo models of HAR in the guinea-pig-to-rat combination. Hamster ovary cells (CHO) and a line of porcine aortic endothelial cells (PAEC11) were transfected with HA complementary DNA (cDNA).The pattern of degradation of rat and human C3 by HA was different (multiple bands lower than 40 kDa) from the physiologic pattern observed after spontaneous degradation of rat C3 or physiologic activation of human C3. The CH50 activity in serum was significantly lower in rats treated with 3.2 mg HA/kg than in untreated rats (45 +/- 16 U/ml vs. 700 +/- 63 U/ml, P0.05). Sera from rats injected with 3.2 mg/kg of HA were less effective in lysing guinea-pig endothelial cells (12 +/- 7%) than normal rat sera (79 +/- 3%; P0.001). Ex vivo, guinea-pig hearts perfused by rat serum supplemented with HA survived longer than those perfused by non-treated serum (210 +/- 34 and 154 +/- 71 min, respectively; P0.05). In vivo, guinea-pig hearts transplanted into HA treated rats survived longer than in non-treated rats (27 +/- 5 min vs. 13 +/- 4 min; P0.001). In the presence of human serum, smaller amounts of C6 and C5b-9 were deposited onto HA-transfected CHO cells than onto control cells. The mHA-PAEC11 cells were significantly more resistant to lysis by human C than control PAEC11 cells.These data suggest that transgenic HA could be used to prevent hyperacute xenogeneic rejection.

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 alpha-subunit (GP alpha), hCG beta-subunit (free hCG beta), 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 pancreatic NET, was found in one patient. Genetic screening of the MEN1 gene was performed in five of the six patients with two components of the MEN1 syndrome. A nonsense mutation (Arg108stop) was identified in the tumor of one patient. Elevated NSE, 5-HIAA, CT, GP alpha, free hCG beta, SMS, and nonsuppressible UFC were found in 47%, 46%, 14%, 19%, 12%, 3%, and 6% of NET patients, respectively. Production of CT, GP alpha, and free hCG beta was highly related to the primary site: all but two of these secretions originated in foregut NET. 5-HIAA secretion was found in 27% of foregut-derived and 85% of midgut-derived NET. In conclusion, MEN1 is a rare event in patients presenting with apparently sporadic NET. It occurred mainly in foregut NET and should be screened for by serum calcium and PTH-(1-84) measurements. Routine hormonal measurements should depend on the primary site. NSE, 5-HIAA, CT, and alphaGP should be routinely measured in foregut-derived NET; only serum NSE and 5-HIAA measurements are recommended in midgut-derived NET.

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.

Human chorionic gonadotropin (hCG) is a glycoprotein composed of two subunits, alpha and beta, linked together by a covalent bond. Ectopic production of hCG has been described in various histological types of cancer. Actually, these malignant tumors predominantly secrete the free beta subunit (hCG beta) and not hCG. Production of free hCG beta is especially found in patients with bladder, pancreas, uterine and lung tumors. In patients with neuroendocrine tumors, serum levels of free hCG beta are higher in gastrointestinal-pancreatic and lung tumors. The significance of ectopic production of hCG beta--epiphenomena or intrinsic biological role--remains unknown. Several reports on the similar structure of hCG beta and certain growth factors suggest that free hCG beta could have an effect on cell proliferation. Increased serum levels of the free alpha subunit are found mainly in patients with neuroendocrine tumors localized in the gut or lung. Serum levels may also be raised in patients with a pituitary tumor, but such production is often associated with a rise in other pituitary hormones. The free alpha subunit plays a role in embryon development and would stimulate production of prolactin by decidual cells. The free alpha subunit may also play a role in tumor growth.

The Rho family of small GTP-binding proteins is one of the three subgroups which, together with the Ras and Rab families, constitute the Ras-related superfamily. The Rho subgroup contains at least seven highly homologous members including 4 Rho proteins (RhoA, RhoC, RhoB, and RhoG), the Rac1 and Rac2 proteins, and CDC42Hs, which are involved in various aspects of cytoskeleton organisation and cell polarity. We have raised antibodies to individual members of the Rho family, and we report here the characterization of a monoclonal antibody (26C4) specific for RhoA. When used in Western blot experiments, the 26C4 antibody recognizes the recombinant RhoA protein but not the almost identical RhoC or the RhoG, Rac and CDC42Hs proteins. Furthermore the 26C4 antibody identifies the natural RhoA protein in human lymphocyte cell extracts and was used to study the level of RhoA expression in several lymphoblastoid cell lines, and its association with the cell membrane.

The diagnostic value of elevated human chorionic gonadotropin (hCG) and its free alpha (hCG alpha) and beta (hCG beta) subunit serum levels as specific tumor markers for nongonadal malignancies is controversial. In the present report, different monoclonal based immunoradiometric assays specific for hCG and its free hCG alpha and hCG beta subunits have been used to reevaluate the presence of these molecules in the serum of patients with a wide variety of tumors. Serum samples from patients with newly diagnosed, persistent, or recurrent malignancies of either known (n = 717) or unknown (n = 32) primary site, healthy blood donors (n = 309), and nonmalignant disease controls (n = 86) were studied using four highly specific and sensitive monoclonal based immunoradiometric assays to hCG and its free subunits. Low level hCG elevations (less than 1000 pg/ml) were found to be common in cancer patients, normal subjects, and disease controls. However, serum levels greater than 1000 pg/ml were highly diagnostic of gonadal tumors and specifically identified nonseminomatous testicular tumors. Significant serum elevations of free hCG alpha subunit (as high as 3000 pg/ml) were found in approximately 96% of cancer patients, normal individuals, and disease controls. In contrast, free hCG beta subunit levels (greater than or equal to 100 pg/ml) were detected in 70 and 50% of patients with nonseminomatous and seminomatous testicular cancers, respectively, and in 47% of bladder, 32% of pancreatic, and 30% of cervical carcinomas. All normal subjects and disease controls had free hCG beta levels less than 100 pg/ml. Thus, the detection of the free hCG beta subunit in serum of nonpregnant subjects was highly diagnostic of malignancy in general and specifically defines a subgroup of aggressive nongonadal malignancies.

Introduction Un taux eleve d’antigene carcino-embryonique (ACE) chez les patients atteints de cancer colorectal (CCR) est generalement considere comme un facteur pronostique pejoratif. Cependant, le taux d’ACE reste un facteur pronostique secondaire dans la plupart des etudes. Le but de ce travail etait d’analyser l’extension de la maladie metastastique, l’efficacite des traitements et la survie lorsque le taux sanguin d’ACE etait superieur a 3 000 ng/mL. Patients et Methodes Entre janvier 2000 et janvier 2007, tous les patients (pts) atteints d’un CCR avec un taux sanguin d’ACE superieur a 3 000 ng/mL, ont ete retrospectivement selectionnes. Le seuil de 3 000 ng/mL etait choisi de facon arbitraire. La survie etait calculee an fonction de la date a laquelle le seuil de 3 000 ng/mL etait atteint. Resultats Quarante huit patients avaient un taux sanguin d’ACE superieur a 3 000 ng/mL, avec des extremes allant de 3 002 a 52 106 ng/mL. Chez 38 pts (gp A), l’ACE atteignait 3 000 au cours du traitement, alors que chez 10 pts, l’ACE etait de plus de 3 000 au moment du diagnostic. Tous les pts avaient une maladie metastatique. Les pts du gp A avaient significativement plus de sites metastatiques que les pts du gp B ; respectivement 1 site, 13 pts vs 7 ; 2 sites, 22 ps vs 3 ; 3 sites, 3 pts vs 0 (p = 0,04). Tous les pts du gp B avaient des metastases hepatiques, isolees chez 7 pts. La survie mediane etait de 7 mois et la survie globale a 1 an etait de 34 %, et de 0 a 5 ans. En analyse univariee, 4 facteurs avaient un impact pronostique positif : un nombre de lignes de chimiotherapie ≥ 2 (p = 0,03), l’administration de therapies ciblees (bevacizumab ou cetuximab) (p = 0,002), une tumeur primitive localisee au rectum (p = 0,01), un taux d’ACE > 3000 au moment du diagnostic et non atteint durant le traitement. Conclusion Un taux sanguin d’ACE superieur a 3 000 ng/ mL est synonyme de maladie metastastique et de mauvais pronostic. Cependant, un sous-groupe de patients atteints de metastases hepatiques isolees avec un taux d’ACE superieur a 3000 ng/mL au moment du diagnostic aurait un pronostic meilleur et pourrait beneficier de traitement plus specifique.

We describe a biodistribution study using a radiolabelled anti-tumor murine monoclonal antibody F(ab')2 in XF-8 in 6 patients with colorectal carcinoma. One patient had an isolated rectal carcinoma. Five patients had hepatic metastasis of colorectal carcinoma. Among them three had also the primary colorectal carcinoma. Double-label method using simultaneous injection of 131-Iodine monoclonal antibody XF-8 F(ab')2 and 125-Iodine control monoclonal antibody was performed to evaluate the specificity of monoclonal antibody localization in the tumor. Each patient received 4-8 mCi of 131-I XF-8 F(ab')2 infused at doses 0.5-5 mg. Simultaneously 200 microCi of 125-I non relevant antibody were infused. Before each administration, immunoreactivity of antibodies preparations was checked before labelling by immunohistological methods and, after labelling, by cell-binding tests. In biodistribution studies, patients were scanned with a scintillation camera each day since the infusing day to the surgery day (3-6 days). To study specificity of XF-8 F(ab')2 antibody localization we measured by means of scintillation counting in tumors and normal tissues recovered after surgery. In vitro immunoreactivity of monoclonal antibody batches was safe. All our data in vivo are negative but correlated. We concluded that criteria for screening the monoclonal antibodies considered for detection of human tumors are not really known.

Calcitonin (CT) is a small hypocalcemic peptide hormone secreted by thyroid C cells [14, 35]. Its physiological role, still a matter of discussion, appears to be that of a hypocalcemic hormone. The ability to measure the circulating level of this hormone may be important in understanding the regulation of calcemia; an additional reason for developing reliable methods for CT measurement stems from the demonstration of its usefulness as a tumor marker in the diagnosis and follow-up of medullary carcinoma of the thyroid (MCT) [40], a neoplastic proliferation of C cells [21, 42], which is transmitted in a familial pattern in 25%–30% of cases [38]. The genetic alteration associated with multiple endocrine neoplasia type 2A has recently been identified [25, 37]. Hyperplasia of the C cells is considered to be a pretumoral stage. Screening of subjects from at-risk families is performed by measuring serum CT level. Diagnosis is usually based either on elevation of the basal level or on a dramatic rise after pentagastrin stimulation. The ability to diagnose MCT in early childhood is essential; indeed, if the tumor is diagnosed when the patient is young, it is likely to be small and without metastasis, in which case surgery (total thyroidectomy) is the treatment of choice [16, 17].

We have produced a library of monoclonal antibodies directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus of hCG. Five antibodies, designated FB01, FB02, FB03, FB04, and FB00, were developed and analyzed with respect to affinity and specificity for epitopes on human chorionic gonadotropin (hCG) and beta hCG by enzyme-linked immunoabsorbent and radioimmunoassays (RIA). All monoclonal antibodies demonstrated low affinity constants (approximately 10(-7) liters/mol) compared with those obtained by immunization with native beta hCG. One antibody, namely FB00, bound only to the synthetic peptide, whereas all other monoclonal antibodies recognized either free native beta hCG or both beta hCG and HCG. Antibodies produced against the synthetic peptide did not cross-react with other glycoprotein hormones such as LH, TSH, and FSH. Characterization of the monoclonal antibody-binding sites revealed the presence of at least four separate and distinct epitopes on the last 35 amino acids of beta hCG. Indeed, one epitope recognized by FB01 is located between residues 109 and 118, whereas another antigenic region recognized by FB04 appears to be present on the 109-121 portion of the molecule near or at position 118. One additional antigenic site was localized between residues 118 and 136. Finally, FB00 recognized an epitope located on the last 10 amino acids (136-145) of beta hCG. With the use of such antibodies, two- and three-site monoclonal RIA were developed and employed to detect free beta hCG and hCG in sera of patients with choriocarcinoma. These assays may be useful in the detection of beta hCG- and hCG-producing tumors and subsequent monitoring of patients in response to surgery and/or chemotherapy.

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.

Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.

In order to detect specifically the beta-hCG, we have produced monoclonal antibodies (Mabs), using as immunogens hCG, beta hCG or a totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus. 34 fusion experiments were performed and led to 8 Mabs which presented a high reactivity to 125I beta hCG in the screening test. Mab D1E8 was directed to the 1-115 region of beta hCG and was cross reactive with hLH (greater than 60%). Mabs 702, 1032, and 1211 were directed against three different epitopes located in the 109-145 region and were specific for the beta hCG. The in vivo localization of tumors containing beta hCG ty tomoscintigraphy (SPECT) was evaluated in 5 patients by injecting 131I-Mab D1E8, selected for imaging because of its high affinity to beta hCG (Ka = 1.9 X 10(9)). SPECT was performed 48 h and 96 h after injection: 2 patients with proven tumor sites had positive "immunoscintigraphy" results. One patient, simultaneously injected with 125I non-specific IgG, had a tumor resection: the count ratio between normal and tumoral tissues revealed a low specific uptake. Two and three-site immunoradiometric assays (IRMA) were performed with monoclonal antibodies purified from nude mice ascitic fluids. IRMA were based upon two or three Mabs, with D1E8 on a solid phase and 125I Mab 702 or a mixing of 125I-702 and 125I-1032 as tracer. The specificity of IRMA was demonstrated by the absence of binding with increasing amount of hLH, hFSH and hTSH up to 5000 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

In an attempt to localize topographic domains specific to native human chorionic gonadotropin (hCG), we studied the discontinuous antigenic regions recognized by a monoclonal anti-hCG antibody designated as C8 which binds only to hCG and does not cross-react with either the free hCG-alpha and hCG-beta subunits or other glycoprotein hormones. Using two-site monoclonal immunoradiometric assays (M-IRMAs), we found that C8 antibody and an anti-peptide antibody (FB12) directed to residues 110-116 of hCG-beta did not bind simultaneously to hCG. This observation suggested that C8 binds to residues of hCG-beta included either in the antibody-binding region of FB12 or in close proximity to amino acids 110-116. To further delineate the regions of hCG-beta recognized by C8, we carried out hapten inhibition experiments with synthetic peptides corresponding to various regions of hCG-beta. The peptide corresponding to residues 109-122 and subpeptides (111-122 or 112-122) inhibited the binding of 125I-hCG to C8, whereas weak inhibition was observed with subpeptide 113-122. By studying the binding of C8 to the 1-112 disulfide-bonded part of hCG-beta (hCG-beta core) recombined with hCG-alpha, we were able to confirm that C8 binds to a region including or near to Asp112. M-IRMAs showed that C8 does not bind to the recombinant molecule lacking residues 113-145 of hCG. Taken together, these results indicate that a limited number of residues located on hCG-beta near to Asp112, and most likely the sequence Asp111-Asp112-Pro113, are included in the discontinuous antigenic region recognized by C8. We then attempted to localize residues of hCG-alpha that constitute another part of the determinant which bound to C8. Six synthetic peptides corresponding to various regions of hCG-alpha did not inhibit binding of 125I-hCG to C8. In contrast, M-IRMAs demonstrated that C8 is capable of binding recombinant products composed of the hCG-beta subunit and the alpha subunits from human, equine, and porcine species. These results indicate that C8 recognizes a region of the alpha subunit highly conserved in these three species. Finally, we determined that the discontinuous regions recognized by C8 are partially accessible on the CG/LH-receptor complex.