Stability of immunoreactive beta-core fragment of hCG

The beta core fragment of hCG (beta C-hCG) accounts for a large proportion of hCG immunoreactivity in the urine of pregnant women. It is often increased in the urine of patients with gynecologic tumors and may become an important diagnostic tool in early pregnancy and cancers. Despite the importance of beta C-hCG, little is known about its stability in urine under conditions of differing pH and temperature. This study examined the effect of repeated freeze-thaw cycles; storage at room temperature, 4C, and -20 C over several months; and the effect of alteration of urine pH. The two specific immunoassays for beta C-hCG used do not significantly cross-react with intact hCG or the free alpha subunit. Despite different cross-reactivities of the antibodies to the free beta subunit and higher immunoactivity when the radioimmunoassay was used, there was excellent correlation between the two assays in pregnancy urine. This suggests that there is little free beta subunit in urine from pregnant women. In addition, this study evaluated the stability of intact hCG and beta-hCG under identical conditions. No alterations in their immunoactivities were found under most conditions of storage. It is concluded that, for clinical purposes, beta C-hCG as well as intact hCG and free beta subunit are very stable molecules.     

Development of highly sensitive enzyme immunoassay to measure urinary beta-core fragment in patients with gynecological cancer]

In addition to human chorionic gonadotropin (hCG) and its free subunits, low molecular weight hCG beta-related fragments have been previously demonstrated in pregnancy urine and in the urine of patients with trophoblastic and nontrophoblastic tumors. The urinary beta-core fragment in particular is focused on as a new tumor marker in gynecological malignancies. We developed an EIA for the beta-core fragment using the monoclonal antibody (MoAb229) which specifically recognized the core portion of hCG beta. By measuring with MoAb229-EIA, it was clearly revealed that urine obtained from normal pregnant women as well as from patients with choriocarcinoma and nontrophoblastic ovarian cancer contains a large amount of the beta-core fragment when separated on Sephadex G-100. We conclude that our MoAb229-EIA is a useful tool to use in detecting the beta-core fragment, a new tumor marker, in the urine of gynecological cancer patients.     

Maternal urine beta-core hCG fragment level and small for gestational age neonates

OBJECTIVE: To determine whether second-trimester urine beta-core fragments of hCG predict small for gestational age (SGA) neonates. METHODS: Spot urine beta-core levels were measured in 733 nonhypertensive women with singleton pregnancies who presented for amniocentesis and had karyotypically normal fetuses. The beta-core level was standardized to urine creatinine and expressed as multiples of the median. The area under a receiver operating characteristics curve was used to determine the screening efficiency of the urine analyte for prediction of small for gestational age (SGA) births. In a subgroup of cases, serum markers (alpha-fetoprotein [AFP], hCG, and unconjugated estriol) were compared using stepwise regression analysis to urine beta-core fragment for SGA prediction. RESULTS: There were 23 (3.0%) SGA neonates. The mean +/- standard deviation (SD) gestation at urine collection was 16.4 +/- 1.3 weeks and collection to delivery interval was 23.0 +/- 2.2 weeks. Mean beta-core (+/- SD) fragment levels were significantly higher in those who later had SGA infants compared with appropriately grown infants (2982.8 ng/mg creatinine versus 1447.4 ng/mg creatinine, P <.001). Stepwise logistic regression found that urine beta-core fragment and serum AFP were the only significant predictors of SGA, with statistically significant chi(2) values (P <.001 and P =.038, respectively). The urine analyte was significantly superior. Second-trimester urine beta-core fragment had a 78.3% sensitivity and 70% specificity for SGA prediction. Exclusion of preeclamptic cases resulted in a sensitivity of 84.2% and a specificity of 71.2%. CONCLUSION: Second-trimester elevated maternal urine beta-core fragment of hCG predicted SGA infants, and was superior to other serum analytes in that prediction.     

Problems in the use of urinary hCG-beta-core as a tumor marker in gynecologic cancer

Urinary human chorionic gonadotrophin beta-core (hCG-βC) was detected in 55–77% of gynecologic malignancies. The use of spot and early morning urine hCG-βC as a tumor marker was explored with regard to the stability of the hCG-βC level in serial spot urine samples collected within 24 hours and in early morning urine collected over 3 days. Thirteen patients with gynecologic malignancies were asked, before treatment, to collect serial urine samples voided within 24 hours. Nine of these 13 patients were also asked to save early morning urine for 2–3 consecutive days. Their urine was assayed for creatinine and hCG-βC using an immunoradiometric assay. Variation of urine concentration was corrected by using the hCG-βC/creatinine (βC/Cr) ratio expressed in pmolg⁻¹. Wide fluctuations of βC/Cr ratios were found both in the serial spot urine within 24 hours and in early morning urine within 3 days. Eight per cent of the patients had one or more spot hCG-βC level double or half the median of their own serial urine samples. Neither spot urine nor early morning urine hCG-βC were suitable for use as a tumor marker for continuous monitoring because of the large fluctuation in hCG-βC levels. The reason for such a wide fluctuation is not clear.     

Following metastatic placental site trophoblastic tumor with urine beta-core fragment.

OBJECTIVE: We document a case with metastatic placental site trophoblastic tumor in a 47-year-old postmenopausal women. METHODS: beta-core fragment was measured in urine using the Triton UGP kit. hCG was also measured using the Bayer Immuno-1hCG assay (at Memorial Sloan-Kettering Cancer Center). RESULTS: Over 2 years the patient underwent two courses of chemotherapy and two debulking operations. During this time, hCG levels decreased from 227 to 4.1 mIU/ml. hCG levels were close to the limit of detection (<3 mIU/ml), indicating complete or near-complete regression of disease. At this point urine beta-core fragment levels were determined. High levels were detected 7.9 fmol/ml, consistent with the continued existence of tumor (>1.9 fmol/ml). High-dose chemotherapy (CEM) was started with stem cell harvesting. In the following weeks hCG levels failed to identify the tumor (4.1 to <3 mIU/ml). In the first week (during therapy) beta-core fragment levels increased (12 fmol/ml), and in the following weeks (after therapy) levels regressed to 1.2 fmol/ml. CONCLUSION: Urine beta-core fragment may be a useful tumor maker when serum hCG levels are near to or below the limit of detection.     

Urinary mesothelin provides greater sensitivity for early stage ovarian cancer than serum mesothelin, urinary hCG free beta subunit and urinary hCG beta core fragment

OBJECTIVES: Early detection of ovarian cancer should improve overall survival. Multiple serum markers have been evaluated as possible tests to detect early stage disease, but few urine markers have been studied. Mesothelin has been detected in serum from patients with ovarian cancer, but has not been previously reported in urine. METHODS: Mesothelin was assayed in the serum and in the urine from 28 patients with early stage (I/II) invasive epithelial ovarian cancers, 111 with advanced stage (III/IV) invasive disease and 19 with tumors of low malignant potential. Marker values have been compared to those in healthy controls and 115 patients with benign pelvic masses. Thresholds were set to include 95% of mesothelin values for 127 sera and 89 urines from healthy women. Urine values were considered: (1) as assayed; (2) normalized using the ratio of serum to urine creatinine; and (3) normalized using the Cockroft-Gault formula for glomerular filtration rate (GFR). Urines were also assayed for human chorionic gonadotropin (hCG) free beta subunit and beta subunit core fragment and similarly normalized. RESULTS: Optimal sensitivity for early stage disease was obtained when urine mesothelin was normalized using GFR. A greater fraction of patients with early stage disease was detected with the mesothelin urine assay (42%) than with the serum assay (12%). Similarly, 75% of patients with advanced ovarian cancer had elevated mesothelin in urine compared to 48% in serum. Serum and urine levels of mesothelin correlated for early (p=0.02) and late (p<0.001) disease. Urine mesothelin exhibited greater sensitivity for early stage ovarian cancer than did hCG free beta subunit or beta subunit core fragment and complementarity was not observed. CONCLUSION: Urine mesothelin deserves further evaluation as a biomarker for detection of early stage ovarian cancer in combination with other urinary markers.     

Placental alkaline phosphatase as a tumor marker in ovarian cancer

The serum levels of placental alkaline phosphatase were determined with a radioimmunoassay using a polyclonal antibody on 1236 samples from 414 patients with ovarian cancer. The frequencies of elevated enzyme levels for patients with or without evidence of disease were 17.7 and 10.9%, respectively. The true positive rate was highest in serous cystadenocarcinoma, undifferentiated carcinoma, and dysgerminoma. A tendency to an inverse correlation with differentiation was found. Measurement of the enzyme did not give a useful index of stage of disease, tumor burden, or prognosis. The value of the enzyme as an index of successful therapy was limited because half of the patients lost this marker during progression. Further studies of the use of this enzyme as a tumor marker should evaluate the modulation of the placental alkaline phosphatase pattern during the course of the disease and should be based on monoclonal antibodies.     

Urinary gonadotropin fragment, a new tumor marker. III. Use in cervical and vulvar cancers

The efficacy of urinary gonadotropin fragment (UGF) and squamous cell carcinoma antigen (SCC) measurements was examined in the management of cervical and vulvar cancers. Of women with benign gynecologic disease (n = 89) 7%, of women with cervical or vulvar intraepithelial neoplasia (n = 25) 8%, and of those with cervical or vulvar malignancies (n = 60) 47% had elevated UGF levels (greater than 3 fmol/ml). SCC at a cutoff of 2.5 ng/ml had a similar sensitivity, 43%, for the same group of cervical and vulvar malignancies. The populations recognized by SCC and UGF, however, only partially overlapped, so that together UGF and SCC were elevated in 62% of women with malignancies. The sensitivity of both markers was stage dependent, so that UGF and SCC detected 26 and 22%, respectively, of early (stage I or II), and 67 and 40%, respectively, of advanced (stage II or IV) cancers. We monitored the progress of 21 women undergoing therapy for cancer with UGF and SCC measurements. Of the 21, 13 (62%) had true-positive UGF levels and 11 (52%) had true-positive SCC levels when cancer was initially detected. The levels of UGF accurately reflected changing clinical observations in 11 of 21 (52%); those of SCC, in 6 of 21 (29%); and levels of either, in 14 of 21 (67%) cases. Recurrences occurred in 7 of the 21 cases. Rising SCC levels predicted (at an earlier clinic visit) the recurrence in 4 of the 7, and rising UGF levels in 5 of the 7 (includes the 4 detected by SCC) patients. While these numbers for UGF and SCC sensitivity are not ideal, until other markers become available, they are seemingly the best achievable. It is suggested that both UGF and SCC be used to monitor therapy and to detect recurrences of cervical and vulvar cancers.     

Hepatoma-derived growth factor expression as a prognostic marker in cervical cancer

AIM: To examine the association of hepatoma-derived growth factor(HDGF) expression with the prognosis of patients with cervical cancer of the uterus(CC). METHODS: HDGF is a unique nuclear growth factor, and it may play an important role in the development and progression of carcinoma. HDGF expression in 88 CC patients aged 23 to 76 years(median, 54 years) was analyzed by immunohistochemistry. A rabbit polyclonal antibody against the C-terminal amino acids(aa 231-240) of the human HDGF sequence was used as primary antibody at a dilution of 1:5000. This specific anti-HDGF antibody was purified using C-terminal peptide-conjugated Sepharose columns. Staining of endothelial cells in the noncancerous areas of each specimen was used as an internal positive control. Samples with more than 80% of tumor cells showing positive immunoreactivity in both the nucleus and cytoplasm were regarded as HDGF index level 2, more than 80% positive immunoreactivity in either the nucleus or cytoplasm as level 1, and less than 80% in both the nucleus and cytoplasm as level 0. The chisquare test and Fisher's exact probability test were used to examine the relationship between HDGF expression and clinicopathologic parameters, and statistical significance was examined by the log-rank test. Multivariate analysis of factors related to survival was performed using Cox's proportional hazards regression model. Statistical significance was set at P 0.05. RESULTS: The five-year overall survival rate was 82.9%. Fourteen patients died due to tumors, nine of whom had tumor recurrence at 2-21 mo(median, 10 mo) after surgery. Tumor recurrence in five patients was determined at the time of the patients' deaths. Nineteen cases were regarded as HDGF index level 0, 11 as level 1, and 58 as level 2. Patients with level 2 expression showed higher rates of histological classification of keratinized squamous cell carcinomaand adenosquamous carcinoma(44.8% of level 2 patients and 13.3% in levels 0 and 1), deep invasion(p T2-4 in 65.5% of level 2 patients, and 30.0% in levels 0 and 1), the presence of lymphatic invasion(50.0% in level 2, and 20.0% in levels 0 and 1), and the presence of lymph node metastasis(37.9% in level 2, and 6.7% in levels 0 and 1). Patients with an HDGF index of level 2 CC showed poorer 5-year overall survival rates than those with level 0 or 1 CC(74.0% and 100%, respectively, P = 0.0036). Univariate analysis revealed that histological classification(P = 0.04), depth of tumor invasion(P = 0.0001), vascular invasion(P = 0.004), and lymph node metastasis(P = 0.0001) were significant factors affecting overall survival in addition to HDGF expression. Multivariate analysis revealed HDGF expression level and lymph node metastasis as independent prognostic factors for overall survival(P = 0.0148 and P = 0.0197, respectively). The prognostic significance of HDGF was further analyzed in p T1 and p T2-4 patient groups, respectively. Among patients with p T1 CC, one the 39 analyzed patients died during the study, and no difference was observed among patients with HDGF index level 0, 1, or 2 CC. However, prognostic significance of the HDGF index was observed in the p T2-4 patient group, in which the mortality rates of patients with HDGF index level 2 CC and those with level 0 or 1 CC significantly differed(P = 0.0463). CONCLUSION: The HDGF expression level is of prognostic significance in CC.     

Urinary gonadotropin fragment, a new tumor marker: I. Assay development and cancer specificity

Urinary gonadotropin fragment (synonyms: UGF and human chorionic gonadotropin beta-subunit core fragment) is a small peptide which is present in the urines of pregnant women, of those with trophoblast disease and of those with certain nontrophoblastic malignancies. We developed a new UGF assay with improved specificity and then investigated levels in urines of 493 women: 155 healthy and postmenopause, 79 healthy and premenopause, 89 with benign gynecologic disease, and 170 with active gynecological cancer. A UGF cutoff level of greater than 3 fmole/ml was chosen to monitor the progress of patients during and after cancer therapy. Using this cutoff value, UGF specificity and sensitivity for active cancer were 90 and 66%, respectively. Levels exceeded this cutoff in 74% of women with recurrent disease. For screening purposes and for differentiating benign and malignant disease a cut-off of 8 fmol/ml, was indicated. At this higher cutoff specificity and sensitivity for active cancer were 99 and 46%, respectively.     

Evaluation of CA125 as a circulating tumor marker for ovarian cancer

CA125 usefulness was evaluated using sera from healthy persons, pregnant women, and patients with ovarian and other tumors. Since serum CA125 levels significantly depended on sex and age in healthy persons, the original cut-off levels were 40 and 25 U/ml in terms of sex and age. Changes in CA125 levels within 40 U/ml were observed during the menstruation cycle. Elevation of CA125 levels was also observed during the first trimester of pregnancy, but these levels fell below 50 U/ml as pregnancy progressed. Immunostaining of the endometrium with OC125 suggested that ovarian function may play an important role in production of CA125 in early pregnancy and menstruating young women. Elevated levels of CA125 were detected in 33/34 (97%) cases with surgically demonstrated ovarian cancer. The clinical usefulness of CA125 for monitoring the course of ovarian cancer was reconfirmed. Practical application of CA125 proved to be useful for the early detection of ovarian cancer and confirmation of the complete disappearance of any tumor.     

Plasma YKL-40, as a prognostic tumor marker in recurrent ovarian cancer

BACKGROUND: YKL-40, a member of family 18 glycosyl hydrolases, is secreted by cancer cells. The function of YKL-40 in cancer diseases is unknown, but it is a growth factor of connective tissue cells and probably has a role in inflammation and remodeling of the extracellular matrix, a process also involved in metastatic malignant diseases. High serum YKL-40 has been associated with poor prognosis for patients with colorectal and recurrent breast cancer. AIM OF THE STUDY: The purpose of the present study was to examine the prognostic value of plasma YKL-40 in patients presenting with recurring ovarian cancer. METHODS: YKL-40 was determined by ELISA in plasma samples from 73 patients with relapse of ovarian cancer shortly before start of second-line chemotherapy. The endpoint used was death because of ovarian cancer. RESULTS: Plasma YKL-40 was increased in ovarian cancer patients (median 94 micro g/L, range 20-1970 micro g/L) compared with age-matched controls (33 micro g/L, range 20-130 micro g/L) (p < 0.001). Fifty-five per cent of the patients had a plasma YKL-40 level above the upper normal 95th percentile of controls. Patients with high plasma YKL-40 (i.e. > 130 micro g/L or > 160 micro g/L) at the time of relapse had significantly shorter survival than patients with normal levels (respectively p = 0.007 and p = 0.004). Plasma YKL-40 proved to be an independent prognostic factor in a multivariate Cox analysis (YKL-40 > 160 micro g/L; HR = 2.27) (p = 0.006), including serum CA-125 and clinical/histological parameters. CONCLUSION: High plasma YKL-40 is related to short survival in patients with recurrent ovarian cancer.     

Evaluation of the Pyruvate Kinase isoenzyme tumor (Tu M2-PK) as a tumor marker for cervical carcinoma

Various isoforms of the glycolytic enzyme pyruvate kinase are expressed in different cell types. One of these isoforms, Tu M2-PK, is over-expressed in tumor cells and released into body fluids. Plasma determination of Tu M2-PK has been shown to discriminate between benign and malignant lesions. Tu M2-PK was quantitated in the plasma of 50 patients with cervical carcinoma, 10 patients with chronic cervicitis and 10 healthy controls. The concentration of Tu M2-PK was determined by commercial kits using a sandwich enzyme linked immunosorbent assay based on two monoclonal antibodies (clone I and II) specific for Tu M2-PK. The sensitivity of the test for discrimination of malignant from non-malignant condition was 82% with a specificity of 60%. Highly significant statistical difference was found in the means of three groups (P = 0.0002). The present results indicate that Tu M2-PK can be used as a tumor marker in follow-up of patients with cervical carcinoma.     

The molar vesicle fluid contains the beta-core fragment of human chorionic gonadotropin

The human chorionic gonadotropin (hCG) beta-core fragment (beta-CF) is a major molecular form of hCG beta subunit (hCGbeta) immunoreactivity in the urine of pregnant women and patients with trophoblastic disease. The majority of evidence supports the fact that the beta-CF is a degradative product of intact hCG and free hCGbeta in the kidneys. We found a beta-CF-like substance in the fluid of molar vesicles from a patient with complete hydatidiform mole. The molar fluid beta-CF (mbeta-CF) was indistinguishable from the beta-CF in the patient's urine (ubeta-CF) by immunoreactivity and by elution profile on gel chromatography. The binding study to lectins, however, showed that mbeta-CF contains a carbohydrate moiety that differs from that of ubeta-CF.Immunohistochemistry with anti-beta-CF antibody demonstrated a strong immunoreactivity in a large number of macrophages in the molar villous stroma. In vitro incubation of intact hCG with peritoneal macrophages showed a slow increase of intact hCG in the cell cytosol with the appearance of beta-CF-like substance in the cell supernatant. In conclusion, the source of beta-CF in molar fluid is likely to be macrophages existing in the villous stroma. Thus macrophages may ingest intact hCG and act as a local regulator of gonadotropic hormones.     

Loss of Fhit expression as a potential marker of malignant progression in preinvasive squamous cervical cancer

OBJECTIVE: In a previous study using the same cases of squamous cervical neoplasia and microinvasive carcinoma (MICA) we found an association between FHIT gene deletion and infection with high-risk HPV (HR HPV). The purpose of this study was to evaluate Fhit protein expression by immunohistochemistry in order to determine whether FHIT gene deletion or infection with HR HPV correlated with aberrant protein expression and grade of lesion. METHODS: A total of 74 archival LLETZ biopsy cases consisting of 23 cervical intraepithelial neoplasia grade 1 (CIN1), 28 CIN3, and 23 MICA cases were selected for Fhit immunostaining. The results of this study on Fhit immunostaining were analyzed in relation to our previous findings using Epi-Info and SPSS-PC statistical analysis software. RESULTS: Fifty percent (14/28) of CIN3 lesions and 78% (18/23) of MICA lesions had a marked reduction or absence of Fhit protein expression (P = <0.001, strength of association, Cramers' V, 0.632). CIN1 lesions were found to have moderate to strong cytoplasmic expression of Fhit. Seventy percent of cases in this study with reduced/absent Fhit protein expression were also positive for FHIT gene loss of heterozygosity (LOH) (P = 0.04, strength of association, phi, 0.254). A significant statistical relationship was found between Fhit protein expression and HPV 16 infection in combined CIN1, CIN3, and MICA cases (P = <0.001). Eighty-seven percent of cases with reduced/absent Fhit protein expression were positive for HPV 16 (strength of association, phi, 0.552). Ninety percent of HPV 16 and 31 positive cases had reduced/absent Fhit expression. CONCLUSION: Our findings suggest an association between HPV infection and FHIT gene abnormalities raising the possibility of a mechanistic role for the FHIT gene as a cofactor with HPV in triggering the development of cervical cancer.     

Granzyme B as a prognostic marker of cervical intraepithelial neoplasia

Granzyme B (GrB) is a serine protease synthesized in T lympocytes (CTL), released after T-cell activation resulting from exogenous stimulation. With perforin, GrB discharges apoptotic signals to a target cell and therefore constitutes a marker to identify activated CTL. We aimed to quantify GrB expression by immunohistochemistry staining in 12 tissue fragments of cervical carcinoma, 33 cervical intraepithelial neoplasias treated by LLTEZ and nine cervical pieces without disease. Activated cytotoxic lymphocyte mean values (20 HPF-400x) in both epithelial and stromal pars were 7.11 cells in tissue without neoplasia, 33.45 cells in cervical intraepithelial neoplasia and 139.75 cells in carcinoma samples, with a statistical difference between them. Comparative analysis in the CIN group showed an expressive difference between cases with disease recurrence (19.28 cells) and without recurrence (37.26 cells). Thus, the relation between number of activated CTLs found at the moment of treatment and clinical evolution determined in this study, suggest GrB use as a prognostic marker.     

Relevance of gamma-glutamyltransferase--a marker for apoptotic balance--in predicting tumor stage and prognosis in cervical cancer

Introduction

Recent large epidemiologic population-based studies identified gamma-glutamyltransferase (GGT) as a marker for increased cervical cancer incidence. Furthermore, high levels of GGT seem to increase the risk of progression of high-grade cervical dysplasia to invasive carcinoma. Therefore, we evaluated the association between pre-therapeutic serum GGT levels, tumor stage and prognosis in patients with cervical cancer.

Materials and methods

In this multi-center trial, pre-therapeutic GGT levels were examined in 692 patients with cervical cancer. GGT levels were correlated with clinico-pathological parameters. Patients were assigned to previously described GGT risk groups and uni- and multivariable survival analyses were performed.

Results

GGT serum levels were associated with FIGO stage (p < 0.0001) and age (r = 0.2, p < 0.0001) but not with lymph node involvement (p = 0.85), and histological type (p = 0.98). High-risk GGT group affiliation (p = 0.01 and p < 0.0001) was associated with poor disease-free and overall survival in a univariate analysis, but not in a multivariable Cox-regression model (p = 0.59 and p = 0.171). We further investigated the association between prognosis and GGT and observed a linear correlation between GGT and prognosis. Therefore we were not able to identify a clear prognostic cut-off value for GGT in patients with cervical cancer.

Conclusions

High GGT - a marker for apoptosis and cervical cancer risk - is associated with advanced tumor stage in patients with cervical cancer.