Urinary gonadotropin peptide (UGP) in Egyptian patients with benign and advanced malignant urological disease.

Urinary gonadotropin peptide (UGP) levels were determined in urine samples from 450 Egyptian subjects to determine its relative level of expression in benign and malignant urological disease, and normal individuals. The mean UGP level in patients with bladder cancer was 44-fold higher than in patients with benign disease, and 81-fold higher than in normal individuals. At specificities of 95% and 100%, overall sensitivities of 73% and 60%, respectively, were observed for the detection of malignant disease. Mean UGP levels in patients with bladder cancer were significantly correlated with the stage and grade of malignant disease but did not vary significantly when stratified according to histological type of disease, nodal involvement or bilharzial association. UGP could be a potentially useful marker for the differentiation of benign from malignant urological disease.

In Eg-pt. bladder cancer is the most common type of male malignancy. ranking only after breast cancer in females in rate of incidence. The disease is characten'sed bs a predominance of locallv ads anced lesions and a high incidence of squamous cell carcinoma (Khaled. 1993). There is a close relationship betus-een the prevalence of urinanr tract schistosomiasis and the incidence of bladder cancer. A positive history of schistosomiasis or repeated treatment of schistosomiasis with anti-bilharzial drugs are correlated With bladder cancer in 9000 of patients (Mustacchi and Shimnkin. 1958: El-Sebai. 1961: Al-Shukri et al.. 1987. Different tumour markers have been evaluated for detecting Egyptian bladder cancer. With varying results in terms of sensitivitv and specificity (El-Ahmadx et al.. 1991a. 1992a. To date. tissue polypeptide antigen (TPA) has been the most reliable marker and the combined use of carcinoembryonic antigen (CEA) and ferritin with TPA has increased the diagnostic value of TPA in detecting bladder cancer (El-Ahmady. 1988: Halim et al.. 1992. 1993. Urinary levels of human chorionic gonadotropin beta subunit (beta-hCG) have also been evaluated in Egyptian bladder cancer patients and patients with benign urinary tract disorders. This marker was elevated in 60.3% of cancer patients. however 29.7% of patients u ith benign disease were also elevated above the upper limit of the normal control group (Halim et al.. 1994).
Urinar-gonadotropin peptide (UGP). also known as unnary gonadotropin fragment (UGF) and beta-core fragment. is a 10.5 kDa glycoprotein with a primarsequence identical to residues 6-40 and 55-92 of the betasubunit of human chorionic gonadotropin (hCG) (Birken et al.. 1988). The carbohvdrate moieties of UGP differ significantly from hCG. lacking all 0-linked species and retaining only the core mannose. N-acetylglucosamine and fucose residues (Bl-the et al.. 1989: Endo et al.. 1989. UGP is measured in urine and is deri'ved from the degradation of ectopic hCG at multiple locations. including the tissue of origin. the circulation and the kidneys (Cole. 1994). UGP is highls stable in urine and studies with pregnancy urines hase indicated that samples can be stored at 4^C or 25'C for 21 days. or -20C for 6 months. Preservatisves are not required to maintain clinical sample stability (de Medeiros et al.. 1991). UGP is not readily measured in serum owing to its rapid clearance rate from the circulation.
UGP is a major component of pregnancy urine. in which it was first described (Franchimont et al.. 1972: Kato andBraunstein. 1988). It has subsequently been shown to occur in the urine of patients with a variety of non-trophoblastic tumours (Papapetrou et al.. 1980). including colorectal cancer (McGill et al.. 1990). pancreatic and biliary cancer. gastric cancer (Alfthan et al.. 1992) and lung cancer (Yoshimura et al.. 1994). Immunohistochemical studies have demonstrated it to be expressed by a w-ide v-ariety of tumour tissues (Kardana et al.. 1988). To date. most studies have focused on its expression in gynaecological cancers. UGP is expressed in a stage-dependent manner in the urine of patients with cervical cancer (Norman et al.. 1990). endometrial cancer (Nam et al.. l990a). N-ulvar cancer (Nam et al.. 1990b) and ovarian cancer (Cole and Nam. 1989).
The objective of this study wvas to evaluate the expression of UGP in preoperative patients with invasisve bladder cancer and benign urological disease and in normal individuals in order to determine its potential use as a marker in the management of this malignancy.

Patients and methods Patients
The present study included 450 indiv-iduals classified into three groups. The first group included 237 patients with urinary bladder cancer who w-ere admitted to the Egyptian National Cancer Institute. This group consisted of 171 males and 66 females ranging in age from 24 to 78 years. with a mean age of 52 y-ears. Lymph node involvement w-as present in 32 patients and absent in 205 patients. Tumour staging was carried out according to UICC cnrtenra and grading A-as according to an established method (Beahrs et al.. 1988). Histopathological examination of the tumour tissues indicated 134 squamous cell carcinomas. 83 transitional cell carcinomas. ten adenocarcinomas. tw-o verrucous carcinomas. tw-o leiomyosarcomas and six undifferentiated carcinomas. As a function of stage. 14 patients A-ere stage T I and T II. 179 patients w-ere stage T III and 44 patients w-ere stage T IV. When stratified by grade. 41 patients were grade 1. 118 patients A-ere grade 2 and 78 patients were grade 3. Bilharzial ova were identified in 143 tumours and absent in 94 tumours. The second group consisted of 97 patients with benign Correspondence: 0 El-Ahmady. 2 Roshdv Street. Safeer Square.
Heliopolis. Cairo. EgYpt Receised 10 April 1995; revised 2 Januars-1996; accepted 15 Januarn 1996 UtGibun Mi-and IC-t magic ase 0 EI-hady et a urinary tract disease recruited from the urology outpatient clinic, Kasr El-Aini Hospital, and included 90 males and seven females ranging in age from 19 to 63 years, with a mean of 28 years. The benign disease categories included 83 patients with urinary tract bilharziasis and 14 with other benign disorders including benign prostatic hyperplasia, renal stones, varicocele and bladder ulcers. The third group included 116 normal healthy controls who were free of disease as evidenced by clinical and laboratory investigations. This group consisted of 107 males and nine females ranging in age from 20 to 52 years, with a mean age of 26 years, who were recruited from students and workers at Al-Azhar University, Cairo, Egypt. All individuals were requested to collect 24 h urines. Approximately 10 ml of each urine sample was centrifuged at 2000-3000 g for 10 min, and the supernatant was frozen at -80:C until analysed.
UGP values are reported in units of fmol ml-' in the 24 h urine samples. Statistical analyses were performed using JMP software (SAS Institute). Population medians were compared using the Kruskal-Wallis rank-sum test.
Resuls UGP levels were determined in 450 timed 24 h urine samples from normal individuals, subjects with benign urological disease and subjects with invasive bladder cancer. The normal, benign disease control and cancer patient cohorts were predominantly male, consisting of 107 (92%), 90 (93%) and 171 (72%) men respectively. The distribution of UGP values in these subject categories is described in Table I. The mean UGP level in the bladder cancer patients was 4.86 fmol ml-', which differed markedly from the mean value for normal subjects at 0.06 fmol ml-' and 0.11 fmol ml-' for the benign urological disease patients. The median UGP levels in the benign disease and normal populations differed significantly from that of the cancer population (P<0.0001), but did not differ significantly from each other.
In order to evaluate the clinical performance of the UGP assay in distinguishing malignant disease from benign disease and normal individuals in this population, two cut-offs were used. These cut-offs were 0.7 and 1.4 fmol ml-', which were the 95th and 100th centiles of the benign disease population. Using these cut-offs, the epidemiological sensitivity of UGP for detecting bladder cancer was evaluated as a function of various clinical parameters. Table H shows the expression of UGP in 116 normal subjects and 97 patients with benign urological disease. The majority of disease control patients (n = 83, 86%) had benign urinary bilharziasis. Mean UGP levels in the normal and disease control populations were similar and ranged from 0 to 0.13 fmol ml-1. Fewer than 1% of normal individuals and 6% of patients with benign disease had UGP levels exceeding the 0.7 fmol ml-' cut-off. The benign bilharziasis group showed the greatest number of patients exceeding the 0.7 fmolmF1 cut-off at 6.0%. None of the patients exceeded the 1.4 fmol ml-' cut-off. Table IH shows the expression of UGP in bladder cancer patients as a function of various parameters. The mean UGP value for all patients was 4.86 fmol ml-'. As a function of   Table III. A trend of increasing UGP values with advancing stage was observed from 3.22 fmol ml-' for stage T I and T II patients to 4.64 fmol ml-' for stage T Ill patients to 6.24 fmol ml-' for stage T IV patients. Median UGP values were significantly different between the stage T Ill and stage T IV patients (P = 0.05) and between the combined stage Tl and T2 patients and stage T IV patients (P=0.05) but not between the combined stage T I and T II patients and the stage T III patients. Similarly, the percentage of patients exceeding the cut-off levels increased as a function of stage. At the 0.7 fmol ml-' cut-off, 64% of stage T I and T H patients, 71% of patients with stage T III disease and 81% of stage T IV patients exceeded the cut-off. The number of patients exceeding the 1.4 fmol ml-' cut-off followed the same trend but was correspondingly lower, ranging from 57% of state T I and T II patients to 73% of stage T IV patients.
When bladder cancer patients were stratified according to grade of disease (Table HI), mean UGP levels were lowest for grade 1 patients, and higher but similar for grade 2 and 3 patients. Grade 1 patients had a mean UGP level of 2.93 fmol ml-' and grade 2 and 3 patients had mean UGP levels of 5.67 and 4.66 fmol ml-' respectively. Median UGP levels were significantly different between grade 1 and grade 2 patients (P=0.006) but not between grade 2 and 3 patients. Overexpression of UGP values was similar for all grades at a cut-off of 0.7 fmol ml-', with 66% of grade 1 patients and 75% and 73%, respectively, of grade 2 and 3 patients exceeding the cut-off. At the higher cut off of 1.4 fmol ml-', the percentage of patients with grade 1 disease exceeding the cut-off was 44%, which was significantly lower than that for the grade 2 (66%) and grade 3 (59%) patients.
Stratification of bladder cancer patients according to nodal status and the presence of bilharzial ova in the tumour tissue is shown in Table III. For both categories, mean UGP levels in negative and positive cases were virtually identical to each other and to the mean value for all cancer patients, ranging from 4.82 to 4.88 fmol ml-'. Similarly, overexpression rates at both cut-offs were virtually identical to each other and to the value for all cancer patients, ranging from 72 -73% at the 0.7 fmol ml-' cut-off, and 58-62% at the 1.4 fmol ml-' cutoff. Finally, stratification of bladder cancer patients according to gender showed no difference in mean UGP levels (data not shown).

DiKmsion
UGP is a pan-marker and has been demonstrated to be expressed in the urine of patients with a variety of solid tumours. Most studies have focused on evaluating the utility of UGP in the management of malignant gynaecological disease, although significant elevations have been observed in other types of malignancies. This study demonstrated that UGP is also overexpressed in a majority of Egyptian patients with advanced stage bladder cancer. The source of UGP in the urine of patients with malignant disease is the metabolic breakdown of hCG species, predominantly hCG beta subunit, originating in the tumour tissue. This is corroborated by previous reports that have demonstrated the presence of hCG beta subunit in the tissues and circulation of approximately 50% of patients with bladder cancer (Oliver et al., 1988;Marcillac et al., 1992). Other studies have shown that UGP was present in the urine of patients with hCGproducing bladder tumours (Iles et al., 1990). Because UGP is the predominant hCG-derived species in urine, it is the most sensitive marker of hCG immunoreactivity for indicating the presence of malignancy. An additional factor contributing to the high level of UGP overexpression in this population of bladder cancer patients could be the relatively high proportion with advanced disease.
In this study population, UGP was demonstrated to be a sensitive and specific marker for malignancy. UGP was only marginally elevated in samples from normal individuals and in patients with benign urological disease. Mean UGP levels in patients with bladder cancer were 81-fold and 44-fold higher than those in normal individuals and patients with benign disease respectively. At the 95% and 100% specificity Ut . bin mud NW -woFhpc-cisnag s oE-Aiady et aX 1489 levels, overall sensitivities of 73% and 60%, respectively, were observed. A statistically significant increase in median UGP level as a function of stage and grade was observed, but no correlation with histological type, nodal involvement or bilharzial association was demonstrable.
The sensitivity of UGP for detecting malignancy in this population of Egyptian bladder cancer patients was comparable with or better than that of other tumour markers. At specificities of 95% and 100%, sensitivities of 73% and 60% respectively were observed. By comparison, at 95% specificity, urinary squamous cell antigen (SCC antigen), ferritin, CEA and TPA were elevated in 24%, 72%, 62% and 81% respectively, of patients with bladder cancer (El-Abmady et al., 1992a, b;Halim et al., 1992). However, at 100% specificity, the sensitivities of ferritin, CEA and TPA dropped markedly to 34%, 23% and 34% respectively.
The UGP cut-offs used in this study are lower than those used in other studies reported in the literature. This could be due to several factors. First, the levels of UGP in cancer patients could be expected to vary according to tumour type. Second, the populations in this study were predominantly male. Earlier studies have shown that the normal range of UGP is measurably higher in post-menopausal women compared with males and premenopausal women (Lee et al., 1991). Finally, this study used 24 urines for UGP determination and the majority of studies reported in the literature with this marker use spot urines, usually corrected for creatinine. Because spot or early-morning spot urines are more readily obtainable than timed 24 h urines, future studies will evaluate the correlation between 24 h urines and spot urines corrected for creatinine.
Owing to its high sensitivity for detecting individuals with malignant disease at a cut-off at which no false-positives were observed in patients with benign urological disease, the clinical value of UGP could be for the differential diagnosis of these patients, particularly in high-risk populations. The application of this marker for this use needs to be evaluated in further studies.
The use of UGP is facilitated by the fact that it is a highly stable marker that is measurable in urine, which is a readily obtained and non-invasive sample. Future studies will focus on evaluating UGP expression in early stage disease, as well as for monitoring and detecting recurrent disease.