CA2234253A1 - Determination and monitoring of bladder cancer - Google Patents

Abstract

A test method for determination of the presence of bladder cancer by determination of the urinary gonadotropin peptide (UGP) levels in no-blood body fluids is described. The method can also be used to monitor the course of bladder cancer and its treatment and the reoccurrance of the disease.

Description

-CA 02234253 l998-04-07 W O 97/14037 PCTnB96/01083 r)FTFRMT~ATIoN AND MONITOPcTNG OF RT AT)nFR CANCFR

Field of the Invention The present invention is directed to methods of detPrmining the presence or absence of bladder cancer in individuals as well as methods of monitoring the effectiveness of the methodology for treating bladder cancer and for monitoring the course of bladder cancer in the body of an individual. Urinary gonadotropin peptide, a core fragment of the beta subunit of human chorionic gonadotropin (hCG) is used as the marker to distinguish bladder cancer as opposed to urogenital benign disease or normalcy and to allow monilo,illg of the course of bladder cancer before or after trP~tment of the cancer.

R~f k~rolm(1 of the InvPntion Bladder cancer has a high incidence throughout the world and in some countries as, for example Egypt, it is the most common type of male m~lign~ncy; and in females, ranks only after breast cancer in rate of incidence. The ~ e~e, as known in the art, is characteri7P~l in the Mideast and Far East by a high predolllhlallce of Iocally advanced lesions, and a high incidence of squamous cell carcinoma, Khaled, H.M. I993, The Cancer J., 6,~5-71 Bladder Cancer and Bilharziasis Today. In SUBSTITUTE SHEET (RULE 26) W O 97/14037 PCT~B96/01083 in~ tri;qli7~cl countries, the predon,i~ l form of bladder cancer is transitional cell carcinoma, and 70% of these cases are superficial, noninvasive types.
Thus, the pathology of bladder cancer can vary as a function of the specific population, and regardless of the histologic type, the main prognostic factors are s stage, grade, and spread of the turnor to tissues outside the bladder. Despite the differences in the natural history of bladder cancer as a function of geography and/or population, tumor markers can be used to aid in the management of ~lis~se At least initially, for each population, the range of values of the tumor marker for normal individuals and patients with benign disease must be ~let~rmin~cl in order to allow 10 determination of a cutoff that distinguishes patients with m~lign~ncies. Generally, a cutoff value for a tumor marker below which 95 percent of normal and benign subjects fall is chosen as the dividing point between normalcy and m~lign~ncy. This 95 percent confidence interval is useful for comparson of the sensitivity of various tumor m~rk~r.s in ~l~tecting m~lign~nt disease.
A variety of tumor m~rk~rs have been evaluated for detecting bladder cancer with varying results and terms of sensitivity and specificity. Tissue polypeptide antigen (TPA) has been one of the most reliable markers and along with the use of carcinoembryonic antigen (CEA) and ferritin with TPA has increased the ~izlgnostic value of TPA in ~etloctin~ bladder cancer. Very recently, urinary levels of human chorionic gonadotropin beta subunit (beta-hCG) have been evaluated in Egyptian bladder cancer patients and patients with benign urinary tract disorders. This marker was elevated in 60.3% of cancer p~fient~ however, 29.7% of patients with benign disease were also elevated above the limits of the normal control group and thus prior studies did not dett?rrnin~ that beta-hCG would be useful as a method of det~?rrnining or monitoring of bladder cancer in individuals. In another study, elevated levels of serum beta-hCG have been shown to occur in 47% of patients with tr~n~itions~l cell bladder cancer (I. Marcillac et al., Free Human Chorionic Gonadotropin beta subunit in Gonadal and Nongonadal Neoplasms. Cancer Research, 52, 3901-3907, 1992). In another study, human chorionic gonadotropin, beta hCG, and beta-core fragment were 3 o demonstrated to be the predolnindlll species present in the serum and urineof several SUBSTITUTE SHEET (RULE 26 CA 022342~3 1998-04-07 ..
.

patients with metastatic transitional cell carcinoma (R.K.Iles et al., Composition of intact hormone and free subunits in the human chorionic gonadotropin-like material found in serum and urine of patients with carcinoma of the bladder. Clinical Endocrinology, 32, 355-364, 1990).
s U.S. Patent 5,356,~17 issued October 18,1994, entitled METHODS FOR
DETECTING THE ONSET, PROGRESSION, AND REGRESSION OF
GYNECOLOGICAL CANCERS, suggested the use of human chorionic gonadokopin beta-subunit core fragment (which is different from HCG) for detecting the progression or regression of gynecologieal cancers in females. Thus, this marker is 0 known as a marker for certain gyneeological cancers and is known for gynecological cancer detection in women by monitoring of non-blood body fluids such as urine. It is often not useLas a marker for det~rrninin~ cancer in some eases beeause the levels determined from blood body fluids are not sufficient for accurate dete~nin~tions. The marker is known to be a major component of pregnancy urine and to occur in the urine of patients with a variety of non-trophoblastic tumors including colorectal cancer, pancreatic and biliary cancer, gastric cancer and lung cancer. Studies have demonstrated it to be expressed by a wide variety of tumor tissues. The marker is expressed in a stage dependent manner in the urine of patients with cervical cancer, endometrial cancer, vulvar cancer and ovarian cancer.
Nishimura et al., Expression and Secretion of the beta Subunit of Human Gonadotropin by Bladder Carcinoma in Vivo and in Vitro. Cancer Research, 55, 1479 - 1484, 1995, demonstrated the secretion of hCG and beta hCG in tissue culture fluid by cell lines, however, UGP was not detected. UGP was detected in the urine of rodents with transplanted tumor cells. Chromatographic traces indicated a similarity between the hCG/beta hCG/UGP profiles in transplanted rodents and a single humansubject. However, there was no demonstration of any potential utility of UGP as a turnor marker based on these experiments, particularly in hllm~n.s.

A~lENDEO SHEET

W O 97114037 PCT~B96/01083 non-blood body fluids, including urine with minimllm inconvenience and with goodaccuracy. ,~
According to the invention, a test method of ~i~tç. ..~ g presence or absence of bladder cancer in an individual comprises obtaining a non-blood body fluid sample s from an individual and then Cl~ p the urinary gonadotropin peptide (UGP) levels in the body fluid of the sample with elevated levels above normal indicating a cancer of the bladder and normal levels indicating the absence of cancer. Preferably, the body fluid is urine and convçnticn~l assay techniques are used to detect andquantify the amounts of urinary gonadotropin peptide (UGP).
o In another method of this invention, an individual is monitored for theeffectiveness of the methodology for treating bladder cancer in the body of the individual known to have bladder cancer. According to the method, in a first step, the individual is treated with any known methodology for reducing or treating bladder cancer. In the next step, the UGP level in a non-blood body fluid of the individual is monitored to indicate the measure of success of the trçatm~nt Preferably, prior to the tre~tmPnt for cancer, a non-blood body fluid sarnple is tested to ~(e~ e the first UGP level and then testing is carried out after tre~tment to rl~(~ ",il.r subsequent UGP
levels. A drop in UGP level when measured successively over a period of time after trÇ~tmpnt indicates some degree of success for the trlo~trnpnt A rise in level would 20 indicate the le~,ulr~nce or growth ofthe cancer, while ~ e of normal values would be encouraging for success of the tre~tment In still another method, a person known to have bladder cancer either as a result of the above tests, and/other medical procedures can be monitored for thecourse of the bladder cancer. The ~cu~ ce of bladder cancer after succç~sful tre~tTnent can also be f~tenninl~l UGP levels are ~let~nined from the urine or other non-blood body fluid over a period of time with variations noting the rise and lowering of cancer activity within the body.
It is a feature of this invention that standard assay techniques can be used in known methods to monitor UGP levels. Another feature of this invention includes the establi~hm~nt of a positive level of UGP above which one can distinguish bladder SUBSTITUTE SHEET (RULE 26) CA 02234253 l998-04-07 W O 97/14037 PCTnB96/01083 cancer from benign urological disease which level is preferably a level above 1.4 fmol/ml, but can be as low as above 0.7 fmol/ml. The testing of this invention for UGP levels can be carried out non-evasively with minimum discomfort to the patient or individual being tested. A single test can indicate elevated levels of concern, whereas prolonged testing over a period of time can be used for monitoring the individual and/or monilolil,g the effectiveness of a standard bladder cancer tre~tment The urine can be tested imm~ tely after withdrawal from the body or at s~lkst~nti~
time periods thereafter while still m51int.qinin~ good sensitivity and accuracy of the test.

Rrief Descriptio}l of the nrawir~
The above and other objects, advantages of the present invention will be better understood from a reading of the following specification in conjunction with theaccoll~l5~ing drawing in which the Figure illustrates the distribution of UGP values in normal subjects, patientC with benign urological ~lice~ce, and patients with bladder cancer. The number of subjects and mean UGP level (fmol/ml) for each group are indicated. The 95% confi~lPn~e intervals for each population mean are indicated by the diamonds.

nescription of Plcrc.-~d Fmbo~liment~
The methods of this invention call for the testing of UGP as a marker for the presence of absence of bladder cancer in a living m~mmAI, preferably man or :~nimzll~
UGP or urinary gonadotropin peptide is also known as urinary gonadotropin ~gment(UGF), human chorionic gonadotropin beta-subunit core fragment and beta-core fr~m~ont UGP is a 10.5 kilodalton glyc~ tein with a primary sequence i-l~ntic~l to residues 6-40 and 52-92 of the beta-subunit of human chorionic gonadotropin ~hCG) as reported by Birken, S. et. al Endocrinol. 123, 572-583 (1988), The Structure of 3 o Human Chorionic Gonadotropin Beta Core Fragrnent from Pregnancy Urine. As is SUBSTITUTE SHEET (RULE 26) W O 97/14037 PCT~B96/01083 known, the carbohydrate moieties of UGP differ ~i~nifi~ntly from hCG, lacking all O-linked species, and ~ only the core mannose, N-Acetylglucosamine, and fucose residues Blithe, DL, et. al, Endocrinol. 125, 2267-2272 (1989), Carbohydrate Composition of Beta Core . As used herein, UGP shall mean urinary gonadotropin s polypeptide as described above.
UGP is measured in urine and can be measured in non-blood body fluids.
Urine is ~Icf~llcd because of its ease of collection in a non-invasive technique in the body. UGP is highly stable in urine, and studies with pregnancy urine have indicated that samples can be stored at 4~ C or 25~ C for 21 days, or at -20~C for six months.
o Preservatives are not required to m~int~in clinical sample stability. UGP is not readily measured in serwn due to its rapid clearance rate from the circulation. It is a major component of pregnancy urine and when pre~ individuals are tested, the test may not be accurate for ~ete~ ion of bladder cancer unless pregnancy dek . Ill;~ ions are also carried out. If pregnancy is detecte~l the instant test cannot be used as an indicator of UGP.
If elevated UGP were found, up until now there was only a concern for identifying gynecological cancers. However, now, if elevated UGP is found, the physician should investigate the presence of both gynecological cancers and bladder cancer.
The tests of this invention are sufficient to evaluate the ~ cssion of UGP in preoperative and postoperative patients with invasive bladder cancer and/or benign urogenital disease and in normal individuals in order to ~letçrmine levels of UGP for use in the management of m~ n~ncy if present.
The testing to ~et~rmine the UGP levels can be by conventional assay 25 techniques. As an example, one assay can involve the dete~nin~tion of levels of UGP
by det~tTnining the levels of hCG beta fragment and beta subwlit and then testing for the C-termin~l peptide (which is not present on the fr~m~nt). A prcr~.,ed assay involves the use of a monoclonal or polyclonal antibody that recognizes any of hCG
beta-fragment or beta-subunit. Known assays useful in this invention include Triton~

SUBSTITUTE SHEE T (RULE 26) , CA 022342F,3 l998-04-07 WO 97/14037 PCT/Is96/01083 UGP EIA kits available from CIBA Corning Diagnostic Corp. of Al~m~ California for the testing and ~ Je mea~urell.ent of UGP in urine.
The Triton~ UGP EIA kit is a two-site enzyme immllno~es~y utili7inFr a specific monoclonal capture antibody and an enzyme-label polyclonal antibody 5 directed towards different antigenic sites on the molecule.
Polystyrene tubes coated with mouse anti-UGP are incubated with urine specimens or the ~p.u~l;ate Calibrator or Control. During this incubation, the UGP
molecules present in the specimen, calibrator and control are bound by the antibody onto the solid phase. Unbound m~teri~l~ present in the specimen are removed by 10 washing ofthe tubes. In the second step, polyclonal anti-UGP Conjugated with horseradish peroxidase is added to the tube. If UGP molecules are present in thespecimen, the Anti-UGP Conjugate is bound to the UGP on the tubes. Unbound conjugate is removed by tube washing. The tubes are next incllb~te~1 with a TMB
Substrate Solution (hydrogen peroxide and 3,3',5,5'-tetramethylben7i~1in~) to develop a color which is a measure of the amount of bound Anti-UGP Conjugate. The hllellsiLy of the color developed is read with a ~e.;llopl1otometer set at 450 nm. Color hlL~ iLy is p~u~olLional to the concentration of UGP in the specimen, within theworking range of the assay. A Calibration Curve is obtained by plotting the UGP
concentration of the Calibrators versus the absorbance. The UGP concentration of the 20 specimen and Control, run concurrently with the Calibrators, can be ~eterrninecl from the Calibration Curve. Since the con-Pntr~tion of urine analytes can vary between samples, ~ aLi~ e levels in the urine can be used to compensate for this variation. In this example, the UGP values are divided by their respective cre~tinine values to give norn-~li7~ values (fmol UGP/mg or mmol ~ile,.~ e). Altt?m~tively, 24 hour urine 25 sarnples can be used or urines can be nor~n~li7~1 by other factors, such as specific gravity.
In a specific exarnple of the present invention, 450 individuals were classifiedinto three groups and testing was carried out to determine presence or absence of bladder cancer.

SUBSTITUTE SHEET (RULE 26) CA 02234253 l998-04-07 W O 97/14037 PCT~B96/01083 The present study included 450 individuals classified into three groups. The first group included 237 patients with urinary bladder cancer that were admitted to the Egyptian National Cancer Institute. This group consisted of 171 males and 66 females ranging in age from 24 to 70 years. Lymph node involvement was present in 32 patients, and absent in 205 patients. Histopathological ~min~tion of the tumor tissues indicated 134 squarnous cell carcinomas, 83 transitional cell carcinomas, 10 adenocarcinomas, 2 verrucous carcinomas, 2 leiomyosarcomas, and 6 nn~ e~ ted carcinomas. As a function of stage 14 patients were stage T I and T II, 179 patients were stage T III, and 44 patients were stage T IV. When stratified by grade, 41 patients were grade 1, 118 patients were grade 2, and 78 patients were grade 3.
Bilh~r7izll ova were identified in 143 tumors, and absent in 94 tumors. Staging and grading were conf~ te~ according to the established TNM and WHO systems, respectively.
The second group consisted of 97 patients with benign urinary tract disease recruited from the urology outpatient clinic, Kasr El-Aini Hospital, Egypt, and included 90 males and 7 females ranging in age from 20 to 63 years. The benign disease c~legolies includes 83 patients with urinary tract bilh~r7i~ci~, and 14 with other benign disorders including benign prostatic hyperplasia, renal stones, varicocele, and bladder ulcers. The third group included 1 16 normal healthy controls who were 20 free of disease as evi~ nce~l by clinical and laboratory investigations. This group con~i~te~ of 107 males and 9 fem~les ranging in age from 20 to 52 years that were recruited from stllderltc and workers at Al-Azhar University, Cairo, Egypt.
All individuals were requested to collect 24-hour unne. Apy~ ately 10 ml of each urine sarnple was centrifuged at 2000-3000 x g for 10 minntes~ and the 25 ~uyel-~n~ l was frozen at -80~C until analyzed.
Urine collection was not limited to 24-hour samples. For example, a comparison of UGP levels in morning urine samples and 24-hour samples from 151 females was made and a significant correlation was found between the two types of samples (r=0.934).

SUBSTITUTE SHEFT tRULE 26) WO 97r14037 PCT~B96101083 Urinary gonadotropin peptide (UGP) was ~let~rminPcl in freshly-thawed urine samples. UGP was measured using an enzyme-linked imm-lno~esay (Triton UGP
EIA, Ciba Corning Diagnostics, Alameda, California). The Triton UGP EIA is a double-~letermin~nt enzyme imml-noassay~ which utilizes a monoclonal capture s antibody immobilized on a coated tube, and an affinity-purified polyclonal antibody conjugated with horseradish peroxidase as the dçtecti- n antibody. The assay has a h~ lulll detectable concentration of 0.1 fmol/ml. Recovery of known quantities of UGP spiked into urine samples ranges from 86 to 109%, with a mean of 96%. The intra- and interassay reproducibility range from 4.12% to 4.95%, and 6.07 to 7.85%, lO ,. ~e.;li~ely, over the range of the assay. Pathological urine samples exhibited linear dilution response, with a mean correlation coefficient of 0.999.
The assay is highly specific for UGP, exhibiting the following molar cross-reactivities: human chorionic gonadotropin (hCG, 0.11%), hCG beta subunit (0.043%), hCG alpha subunit (0.009%, human luteinizing horrnone (hLH, 0.001%), hLH beta subunit (0.005%), human thyroid stim~ ting hormone and beta subunit (hTSH and hFSH-beta subunit, <0.001%), and human follicle stim~ tin~ hormone and beta subunit (hFSH and hFSH-beta subunit, <0.001%). The assay has been ~p~ d to elimin~t~ cross-reactivity with fragments derived from luteinizing hormone that are present in urine. The following urinary analytes do not interfere with the assay at levels up to the following concentrations: urea (5 g/dL), uric acid (150 mg!dL), cl~ e (500 mg/dL), creatine (200 mg/dL), vitamin C (500 mg/dL), urobilin (4 mg/dL), glucose (30 mg/dL), and hemoglobin (lO mg/dL). The acceptable pH range of urine samples is from about 5.5 to about 8.5.
Further variations will be ~t;nL to those with oldh.al y skill in the art. The following e~mrles illustrate various aspects of the invention but are not intended to limit its usefulness.

G Fx~nu~le 1 UGP levels were ~let~rtnined in 450 timed 24-hour urine samples from normal 30 individuals, subjects with benign urological ~ e~e, and subjects ~,vith invasive SUBSTITUTE SHEET (RULE 26) CA 022342~3 1998-04-07 W O 97/14037 PCTnB96/01083 bladder cancer. The norm~l, benign disease control, and cancer patient cohorts were predomin~ntly male, conci~ting of 107 (92%), 90 (93%), and 171 (72%) male subjects, respectively. The distribution of UGP values in these subject categories is shown in the Figure. UGP values are reported in units of fmol/ml in the 24-hour urine samples. Statistical analyses were performed using JMP software (SAS Tn~titllte).
Population means were compared using the Tukey-Kramer HSD method. The mean UGP level in the bladder cancer patients was 4.86 frnol/ml, compared with 0.06 fmol/ml in the normal subjects, and 0.1 1 fmol/ml in the benign urological disease patients. The mean UGP levels in these populations were significantly different (p<O.Ol).
In order to evaluate the clinical p~lrollllance of the UGP assay in distinguishing m~lign~nt disease from benign disease and normal individuals in this population, two cutoffs were used. The first cutoff was 0.7 fmol/ml, which was the calculated 95% specificity level based on two standard deviations above the meanUGP level ofthe benign disease population. The second cutoffwas 1.4 fmol/ml, which was the 100% specificity level based on the distribution of IJGP values in the same population. Using these cutoffs, the epidemiological sensitivity of UGP fordetecting bladder cancer was evaluated according to various clinical parameters.Table I shows the ~xlJr~s~ion of UGP in 1 16 normal subjects, and 97 patients with benign urological disease. The majority of disease control patients (N = 83,86%) had benign urinary bilh~r7~ Mean UGP levels in the normal and disease control populations were similar, and ranged from 0 to 0.13 fmol/ml. Less than one percent of normal individuals, and six percent of patients with benign disease had UGP levels excee~1ing the 0.7 fmol/ml cutoff. The benign bilh~t7i~ci~ group showed the ~;,.,ale~l number of patients excee-ling the 0.7 fmoVml cutoff, at 7.2%. None ofthe patients exceeded the 1.4 fmol/ml cutoff.
Table II shows the ~x~.~s~ion of UGP in bladder cancer patients as a function of histologic type of disease. The mean UGP value for all patients was 4.86 fmol/ml.
Patients with squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) had the highest mean UGP levels of 4.84, and 5.40 fmol/ml, le~ecLively. Patients SUBSTITUTE SHEET (RULE 26) CA 022342~3 1998-04-07 W O 97/14037 PCT~B96/01083 with other histologic types of m~lign~nt ~li.ce~e, including adenocarcinoma, undiffert?nti~t~l carcinoma, verrucous carcinoma, and leiomyosarcoma had a mean UGP level of 2.76 fmol/ml. The dirLIences in the mean UGP levels between these histotypes were not statistically significant (p > 0.05). The percent of patients eXceelling both cutoffs was similar for the TCC and SCC patients, for example, 65%
of SCC patients and 71% of TCC patients exceeded the cutoff of 0.7 fmol/ml.
Patients with other histologic types of disease exceeded both cutoffs in 50% of all cases.
Analysis of bladder cancer patients according to stage of disease is shown in Table III. An increase in UGP values from 3.22 fmol/ml for stage T I and T II
patients, to 4.64 fmol/ml for stage T III patients, to 6.24 fmol/ml for stage IV patients was observed. Despite the ~~ ellt trend of increasing mean UGP level with advancing stage, these mean values were not significantly different (p > 0.05).
Similarly, the percent of patients excee~ling the cutoff levels increased as a function of stage. At the 0.7 frnol/ml cutoff, 64% of stage T I and T II patients, 71% of patients with stage T III ~ e~o7 and 81% of stage T IV patients exceerling the cutoff. The number of patients exceeAing the 1.4 fmol/ml cutoff followed the same trend, but was correspondingly lower, ranging from 57% of stage 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 IV), 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. The dirr~-~nces in mean UGP levels were significant only between the grade I and the combination of grade 2 and 3 disease (p < 0.05). Overexpression of UGP values was similar for all grades at a cutoff of 0.7 fmol/ml, with 66% of grade 1 patients, and 75% and 73%, respectively of grade 2 and 3 patients excee~lin~ thecutoff. At the higher cutoff of 1.4 fmol/ml, the p~rce.ll~ge of patients with grade 1 disease exceeding the cutoffwas 44%, which was ~i nific~ntly lower than the grade 2 (66%) and grade 3 (59%) patients.

SUBSTITUTE SHEET (RULE 26) , W O 97114037 PCT~B96/01083 Stratification of bladder cancer patients according to nodal status and the presence of bilh~r7i~l ova in the tumor tissue is shown in Table V. 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, ove~ ssion rates at both cutoffs were virtually identical toeach other and to the value for all cancer patients, ranging from 72-73% at the 0.7 fmol/ml cutoff, and 58-62% for the 1.4 fmol/ml cutoff. Finally, stratification of bladder cancer patients according to gender showed no difference in mean UGP
levels. See data in Table VI.) The above example demonstrated that UGP is also o~ e~ ed in a majority of patients with invasive bladder cancer.
In this study population, UGP was demonstrated to be a sensitive and specific marker for m~ n~ncy. UGP was only marginally elevated in samples from normal individuals, and in patients with benign urogenital disease. Mean UGP levels in patients with bladder cancer were 81 and 44-fold higher than those in normal individuals, and patients with benign disease, l~,~e~,lively. At the 95% and 100%
specificity levels, an overall sensitivity of 73 and 60%, respectively, was observed.
No statistically significant correlation of UGP levels with histologic type, stage, grade, nodal involvement, or bilh~r7i~l association was demonstrable, although a2 0 trend of increasing mean UGP levels with stage of disease was observed.
The sensitivity of UGP for detecting m~lign~nl~y in this population of Egyptian bladder cancer patients was comparable to or better than that of other turnor markers. At a specificities of 95% and 100%, sensitivities of 73% abd 60% were observed, respectively. UGP showed an extremely low positivity in p~ti~ te with 2 5 benign urinary tract disorders.
Due to the extreme difference in UGP levels between patients with benign ~1i.eç~ec7 and bladder cancer patients, UGP is useful for the differential diagnosis of these patients.
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.

CA 022342~3 1998-04-07 W O 97114037 PCTnB96/01083 The above example established a level for differenti~tin~ bladder cancer in the hllm~n~ tested for benign urological ~ e~e~l that is m~ n~nt (or bladder cancer)from benign disease. The levels indicate that levels above 0.07 fmol/mL are significant as a distinguishing point with a level above 1.4 fmol/mL ~lefining a clear-cut distinguishing point of m~ n~nt cancer herein bladder cancer, over benign disease. Of course, when testing women, there is a chance that urine may indicate the presence of cancer other than bladder cancer. Thus, women who, when tested, testpositive, have to be further tested or pretested to rule out other forms of cancer or pregnancy. Biopsy can be used, as can and other known methods for confirming theo absence of other cancers or the presence of bladder cancer. Similarly, in pregnancy, in some cases7 higher levels of UGP may require further testing to be certain of an indication of bladder cancer. The level for differPnti~ting bladder cancer from benign disease may vary between populations, however, the above example indicates the method for applying the use of UGP to dirr~1c1l~ populations, or additionally with other methods of urine collection such as spot urines that require creatinine or other types of norm~li7~tion.
The UGP d~lf ~ ;on can be used as a screening test for bladder cancer .
There is a clear distinction between bladder cancer (i.e., m~ n~ncy) and benign disease.
Additionally, UGP expression is independent of the histologic type of bladder cancer, therefore, it can be used for the detection, monitoring, or screening of any histotype of ~ e~e, especially transitional cell and squamous carcinomas.
In addition to testing for the ~-ese1lce or absence of bladder cancer with a reasonable degree of certainty or as a screening test, the det~nnin~tion of UGP in the urine of males and females can be used to monitor the progression of bladder cancer in an individual known to have bladder cancer. Higher levels of UGP are observed in advanced stages and grades, and stage l and 2 disease can be st~ti~tic~lly dirr~1G..Ii~tçd from stage 3 and 4 disease.
For example, UGP levels can be deterrnin~l in an individual otherwise known 30 to have bladder cancer. The ~1~L~ itl;on of bladder cancer can be made by the test SUBSTITUTE SHEET (RULE 26) W 0 97114037 PCT~B96/01083 of this invention with or without the use of biopsy. Thus, if a first level of UGP at an elevated level is established in a bladder cancer patient, that level can be monitored at 24 hour, 4 day, one week, monthly or other periods to ~let~rminP the progression or regression of the ~liee~ce, with higher levels indicating increased cancer and lower s levels indicating reduced cancer, and normal levels indicating absence of disease.
Norrnal levels would be considered any level below 0.7 fmol/mL or at least below 1.4 fmol/mL in the urine, when measured in 24 hour urine samples. However, the optimum cutoff for distinguishing normal individuals and individuals with benigndisease from individuals with m~lign~n~y may need to be determined for each 10 population that is being studied. Other non-blood body fluids such as interstitial fluids and the like, Iymph fluids or other fluids found in the urinary tracts ofindividuals can also be used for ~l~terminin~ UGP level.
To monitor the progression or regression of disease in an individual, UGP
levels are (let~rrnin~cl using the Triton~) Ciba-Corning kit, as known in the art during lS preselected time periods, as di~ cse~l above. Other assay techniques lltili7in~
monoclonal and/or polyclonal antibodies in sandwich or co~ Lilive assay formats can similarly be used to measure UGP levels. In addition, the patient can be treated with a methodology for reducing bladder cancer. Any known methodology can be used as, for example, X-ray tre~tmt?nt chemical tre~tm~ntc, intravesical chemotherapy, laser therapy, immunotherapy, surgery or the like. After such ke~tment the body of the individual can be monitored for UGP levels by monitoring the urine with the levels found again in~ ting the presence or ~hsen(-e of m~lign~ncy even after the l,e<~ nt Thus, levels above the levels previously discussed wouldindicate malignancy. Declining levels would indicate the cancer's (leclin~, while increasing levels would indicate growth and progression of the cancer. Normal levels would indicate probably recovery of the patient.
The monitoring of the patient after L-eaL~ with a cancer therapy can be accomplished by carrying out 4~ l; ve deterrnin~tion of UGP levels at any selected period desired.

SUBSTITUTE SHEET (RULE 26) W O 97/14037 PCTnB96/01083 F~n~le 2 A second group of patients, including more with early stage bladder cancer, was evaluated for UGP, with the results apl~e~iSlg in Table VII. UGP levels in stage TI and TII disease were ~i~nific~ntly elevated above the normal and control s population and were not significantly different from levels in patients with invasive stage TIII ~lise~e Thus, this data shows that UGP is also a reliable indicator of early stage bladder cancer.

Fx~ml7le 3 In a specific example of monitoring the course of cancer in a patient, a patienthaving bladder cancer at stage T3 is treated by surgery.
The UGP ~ iLZll i ve level in the urine of the patient is determined prior to the treatment at a level of 4.8 fmol/mL. After tre~tment, the patient is monitored at monWy intervals and found to have levels of 0.3 fmol/mL at 1 month, 2 months, and 3 months post surgery, indicating that the patient was free of residual ~ ç~ee In this theoretical example, the patient has ~l~çlinin~ UGP values to normal, in-lir~tin~ success of the treatment and return of the patient to normal, at least for that period of time. Rising or original values would be an indicator of lack of success of the tre~tment However, it should be noted that the q~ e test is indicative for dirf~,lellL
stages of cancer. Thus, there is a correlation that can be made between UGP levels, and stage 1, stage 2, stage 3, stage 4 or other stages of cancer. The various stages of cancer are known in the art and described in "Clinical Oncology", M.D. Abeloffet al., eds., in the chapter titled Management of Specif c Malignancies, 66 Bladder 1422-1433, 1995.

While specific embo~liment~ of the present invention have been described above, many variations are possible. In all cases, the invention comprises the dçtt?nnin~tion of UGP levels, to indicate presence or absence of bladder cancer and SUBSTITUTE SHEET (RULE 26) CA 02234253 l998-04-07 W O 97/14037 PCT~B96/01083 distinguish it from benign urinary disease and/or to monitor tre~tmçnt of patients having bladder cancer.

SUBSTITUTE SHEET (RULE 26) W O 97/14037 PCT~B96/01083 'I'al~lc r I xprcssioll of UGI' in nom1al and control subjects Mcan UGI' Nunlbcr(%) cxccedingcutofr r~angc Category N (fmol/mE) S D. 0.7 fmol/ml, 3.4 fmol/ml, Min. - Max Norrnal ~ I IG 0.06 0 1~ 1 ( 0.9~ o 0 - 0 8 Benign Urinary tract disease b B;Ih~ 3 0.13 0.31 6 (7 2) 0 O - 1.37 Other c 14 0.0 N/A 0 0 0 - 0.0 Total bemgn 97 0 1] 0.29 6 (6 2) 0 0 - 1 37 ' 107 male, 9 female. b 90 male, 7 fiemale. ' Benig~ prostatic hyperplasia, renal stones, varicocele, bladder ulcer.

SUBSTITUTE SHEET (RULE 26) W O 97/14037 PCTnB96/01083 Tal)lc11 E~prcssiol~rlJGl'illpatienis~ bladdcrc~nccr:brc~kdo~JIbvhislolog~

Mean UGI' Numbcr (%) c~;cceding cutoff r~angc Calcgor~ N(fiTnol/mr~) S D. () 7 fn~ol/ml~ 1.4 ~mol/ml Min. - Max.

SCC 134 4.84 5.40 87(65) ~1 (60) 0-20.2 TCC 83 5.40 5 47 59 (71) 52 (63) 0 - ~8.5 Other - 2û 2.76 3 74 10 (50) 10 (50) 0 - 15.1 Total b 237 4.86 5.34 172 (73) 142 (60) 0 - 20.2 A Adenocarcinoma, undifferentiated carcinoma, verrucous carcinoma, leiomyosd~,oll~a b 171 male, 66 female.

SUBSTITUTE SHEET (RULE 26) CA 02234253 l998-04-07 W O 97/14037 PCT~B96/01083 Tablc III E~;prcssion of UGI' in paticnts ~ith bladder canccr: breakdo~l by slagc Mc~n UGP Number (~/O) excccding cutoff R~1gc Category N (fmol/ml,) S D. 0.7 fmol/ml, 1.4 fmol/ml, Min. - Max.

StageTI~TII 14 3.22 3.91 9 (64) 8 (57) 0 - 12.0 Stage T III 179 4.64 5.40 127 (71) 102 (57) 0 - 20.2 Stage T IV 44 6.24 ~.2S 36 (8 1 ) 32 (73) 0 -1 6.5 Total 237 4.86 5.34172 (73) 142 (60) 0 - 20 2 SUBSTITUTE SHEET (RULE 26) lc IV I~xprcssion of UGI' ill paticnts willl bladdcr C~lllCCI: brc;31;down by gradc Mcan UGI' Numbcr (%) cxcecdin,e culoff Rangc Catc~ory N( fmol/mL) S.DØ7 ~mol/ml, 1,4 fmol/ml, Mim - Max.

Gradcl 41 2.93 4.1627(66) 18(44) ()- 16.0 Grade 2 118 5.67 5.72S8 (75) 78 (66) 0 20.2 Gradc 3 78 4.66 5.0457 (733 46(59) 0 - 18.5 To al 237 4.86 5.34172 (73) 142 (60) 0 - 20.2 SUBSTITUTE SHEET (RULE 26) CA 02234253 l998-04-07 W O 97/14037 PCTnB96/01083 I ai~lc v L~:prcSslOIl of UGP in patienis ~'itll ;laddcr callccr: l~rcal~do~ nodal stalus and billlarzial :~ssocialion Mean UGP Numbcr (%~ cxcccdill~, cutofi Ran<~e Catcgor~ N ( fmol/mL) S.D ~).7 fillol/ml~ 1.4 fmol/mT, Min. - Ma~.

Nodal status Ncgativc 205 4 86 5 36 149 (7~) 123 (60) 0 - 20.2 Positive 32 488 5.21 23(?2) 19(59) 0 170 Bilharzial ova in tumor tissue Ncgativc 93 4.83 5.46 67 (72) 58 (62) 0- 19.0 Positive 143 4.82 5.22 104 (73) 83 (58) 0-20.2 Total cancer 237 4.86 5.34 172 (73) 142 (73) 0 - 20.2 SUBSTITUTE SHEET (RULE 26) W O 97/14037 PCTnB96/01083 T:~blc Vl Exprcssion of UGP in patients ~'itll bladder callcer: breakdo~ b)~ ~ender Cate~or~ N Mean a~e Mean UGP (~mol/mL) S.D.

I:emale 66 50 4.91 5.50 Male 171 53 4.~1 5.~3 SUBSTITUTE SHEET (RULE 26) CA 02234253 l998-04-07 W O 97/14037 PCT~B96/01083 Tablc Vll UGP in the differenl stagcs of bl~dder cancer UGP TI 1'11 1 111 TIV I'I + 11 l 111 t IV
(fmol/ml) (n=14) (n=26) (n=188) (n=49) (n=40! (n=237) Mean 3.4~ 3.77 4.71 *6.60 3.64 ~. I
SE 1.36 1.22 0.39 0.78 0.91 0.35 Median 1.63 1.78 2.15 5.85 1.78 2.60 Minimum 0.34 0 0 0 0 0 Maximum 17.6 30 20.2 17 30 20.2 % with un-detectable 21.4 30.8 43.6 63.3 27.5 47.7 levels *Significatly different from T III at p<0.05 SUBSTITUTE SHEET (RULE 26)

Claims (16)

What is claimed is:

1. A test method of determining presence or absence of bladder cancer, said method comprising, obtaining a non-blood body fluid sample from an individual, determining urinary gonadotropin peptide (UGP) levels in said non-blood body fluid sample of said individual with elevated levels above normal indicating a possible cancer of thebladder and normal levels indicating the absence of cancer.

2. A method in accordance with claim 1 wherein said non-blood body fluid is urine.

3. A method in accordance with the method of claim 2 wherein said urine sample is stored at temperatures of up to 37 degrees C for no more than thirty days before said determining step.

4. A method in accordance with the method of claim 2 wherein said urine sample is stored at a temperature of about minus 20 degrees C for one month prior to said determining step without the use of protease inhibitors.

5. A method of determining the presence or absence of bladder cancer in accordance with claim 2 wherein said determining step is carried out by a doubledeterminant enzyme immunoassay or other methodology.

6. A method of determining possible presence or absence of bladder cancer in accordance with claim 2 wherein an individual is first screened to determined the absence of other cancers and pregnancy.

7. A method in accordance with the method of claim 2 wherein said elevated level above normal comprises any level above the 90 - 95 percent confidence interval of the normal population, or population with benign disease that are being evaluated.

8. A method in accordance with the method of claim 2, wherein said elevated level above normal comprises any level above the 100 percent confidenceinterval of the normal population, or population with benign disease that are being evaluated.

9. A method of monitoring the effectiveness of a methodology for treating bladder cancer in an individual known to have or to have had bladder cancer, said method comprising, in a first step, measuring the UGP level and treating said individual with a methodology for reducing bladder cancer, and, in a next step, determining UGP level in a non-blood body fluid of said individual with said UGP level indicating a measure of the success of said treatment.

10. A method of claim 9 in which successive samples are evaluated over time to determine the effectiveness of said methodology.

11. A method in accordance with the method of claim 9, wherein said determining step is carried out by a double determinate enzyme immunoassay.

12. A method in accordance with the method of claim 9, wherein prior to said treatment a non-blood body fluid sample from said individual is tested to determine a first UGP level in said non-blood body fluid, and comparing said next step UGP level with said UGP level determined before said treatment, with the level of UGP after said treatment indicating a measure of success of said methodology.

13. A method in accordance with the method of claim 9 wherein said non-blood body fluid is urine.

14. A method in accordance with the method of claim 11 wherein said non-blood body fluid is urine.

15. A method for monitoring the course of bladder cancer in the body, said method comprising determining the presence or absence of bladder cancer in an individual and monitoring said individual to determine the level of UGP in urine of said individual at successive time periods with rising levels indicating increased cancer incidence or disease recurrence and declining levels indicating decreasedcancer levels in the bladder.

16. A method in accordance with the method of claim 15 wherein said monitoring is carried out in urine samples taken from said individual.