METHODS FOR DETECTING AND FOLLOWING THE COURSE OF CANCER, PREGNANCY AND TROPHOBLASTIC DISEASE

Abstract: A method for detecting and following the course of a cancer, pregnancy or a trophoblastic disease comprising analyzing whole blood or a fluid of whole blood for the presence of serum gonadotropin peptide complex or a component of serum gonadotropin peptide complex, wherein the presence of serum gonadotropin peptide complex or a component thereof is an indication of the presence of a cancer, pregnancy or a trophoblastic disease in a patient from which the blood or fluid of whole blood was obtained.

Description: METHODS FOR DETECTING AND FOLLOWING THE COURSE OF CANCER, PREGNANCY AND TROPHOBLASTIC DISEASE GOVERNMENT RIGHTS This invention was made with United States government support under Grants CA-44131 and CA-53828 from the National Cancer Institute. The United States government thus has certain rights in this invention. BACKGROUND OF THE INVENTION Field of the Invention The present invention concerns methods for detecting and following the course of cancer, pregnancy and trophoblastic disease by analyzing whole blood, plasma or serum for the presence of serum gonadotropin peptide complex or a component thereof. Background Information Trophoblast cells secrete the hormone hCG (human chorioniσ gonadotropin) and a small molecule, namely beta- core fragment ("BCF") (composed of two small peptides that are parts of the hCG molecule) throughout pregnancy. The molecule hCG is the sole marker used for detecting and following the course of pregnancy. While hCG can be detected in pregnancy blood/serum and urine at high levels, the beta core fragment can be detected only in urine. Heretofore, the beta core fragment could not be detected in blood/serum. This has greatly limited the diagnostic SUBSTITUTESHEET applications of the beta core fragment in the management of pregnancy and the detection of ectopic production by cancers. The hCG β-subunit is a polypeptide of 145 amino acids (MW = 22,200) . The β-core fragment is composed of segments of hCG β-subunit (residues 6-40 disulfide-linked to residues 55-92), and is about half the molecular weight (10,300). Two N-linked sugar chains are linked to the β-core fragment at Asn 13 and Asn 30; these are devoid of sialic acid and are of different structures to those at analogous positions on the hCG β-subunit (Birken, S., Armstrong E.G., Gawinowiσz-Kolks, M.A. , Cole, L.A., Agosto, G.M. , Krichevsky, A., Vaitukaitis, J.L. , Canfield, R.E., "Structure of the Human Chorionic Gonadotropin β-subunit Fragment from Pregnancy Urine", Endocrinology. (1988) 123:372-83; Cole, L.A. , Birken, S., "Origin and Occurrence of Human Chorionic Gonadotropin β-subunit Core Fragment", Mol Endocrinol. (1988); 2:825-30; and Blithe, D.L. , Wehmann, R.E. , Nisula, B.C., "Carbohydrate Composition and β-Core", Endocrinology. (1989); 125:2267-72). Recent studies have shown that the β-core fragment in urine is associated with 2 small peptides as a complex (Kardana et al., submitted for publication). High levels of the β-core fragment have been detected in the urine of women with pregnancy, trophoblast disease and certain cancers (Birken et al, supra; Cole et al, supra; Cole LA, Wang Y, Elliott, M.E., Latif, M. , Chambers, T. , Chambers, S., Schwartz, P.E., "Urinary hCG Free β-subunit and β-core Fragment: A New Marker of Gynecologic Cancers", Cancer Res.. (1988) 48:1356-60; Wehmann, R.E., Nisula, B.C., "Characterization of a Discrete Degradation Product of Human Chorionic Gonadotropin β-subunit in Humans", J. Clin SUBSTITUTESHEET Endocrinol Metab.. (1980) 51:101-5; Nam. J, Cole, L. , Chambers, J. , Schwartz, P., "Urinary Gonadotropin Fragment, a New Tumor Marker", Gvnecol Oncol. (1990) 36:383-90; Schroeder, H. , Halter, C. , "Specificity of Human β-choriogonadotropin Assays for the Hormone and for an Immunoreactive Fragment Present in Urine During Normal Pregnancy", Clin Chem. (1983) 29:667-71; Papapetrou, P.D., Nicopoulou, S.C., "The Origin of a Human Chorionic Gonadotropin β-fragment in the Urine of Patients with Cancer", Acta Endocrinol (1986) 112:415-22; Masure, H. , Jaffee, W. , Sickel, M. , Birken, S., Canfield, R. , Vaitukaitis, J. , "Characterization of the Small Molecular Size Urinary Immunoreactive hCG-like Substance Produced by Normal Placenta and by hCG Secreting Neoplasms, J. Clin Endocrinol Metab. (1981) 53:1014-20; Krichevsky, A., Armstrong, E., Schlatterer, J. , Birken, S., O'Connor, J. , Bikel, K. , Silverberg, S., Lustbader, J., Canfield, R, Preparation and Characterization of Antibodies to Urinary Fragment of Human Chorionic Gonadotropin β-subunit". Endocrinology. (1988) 123:584-93; Akar, A.H. , Wehmann, R.E., Blithe, D.L., Blacker, C. , Nisula, B.C., "A Radioimmunoassay for the Core Fragment of the Human Chorionic Gonadotropin β- subunit", J. Clin Endocrinol Metab. f (1988) 66:538-45; Good, A., Ramos-Uribe, M. , Ryan, R.J., Kempers, R.D., "Molecular Forms of Human Chorionic Gonadotropin in Serum, Urine and Placental Extracts", Fertil Steril. (1977) 28:846-50; O'Connor, J., Schlatterer, J. , Birken, S., Krichevsky, A., Armstrong, E., McMahon, D. , Canfield, R, "Development of Highly Sensitive Immunoassays to Measure hCG, its β-subunit, and β-Core Fragment in the Urine: Application to Malignancies", Cancer Res.. (1988) 48:1361- 66) . Using immunohistochemistry, β-core fragment has been detected in trophoblast tissue and in 93% (77/83) of SUBSTITUT different tumor sections (Kardana, A., Taylor M. , Southall, P. , Boxer, G. , Rowan, A. , Bagshawe, K. "Urinary Gonadotropin Peptide-isolation and Purification, and its Immumo- Histochemical Distribution in Normal and Neoplastic Tissues," Br J Cancer. (1988) 58:281-6). While high-levels of β-core fragment have been reported in urine and tissue samples, little or no immunoreactive molecules of the size of β-core fragment have been found in serum (Cole et al. Cancer Res.. (1988), 48:1356-60; Wehmann, R., Blithe, D., Akar, H. , Nisula, B, "Disparity Between β- core Levels in Pregnancy Urine and Serum: Implications for the Origin of Urinary β-core, J. Clin Endocrinol Metab. (1990) 70:371-78 and Alfthan, H. , Stenman, U. , "Pregnancy Serum Contains the β-core Fragment of Human Choriogonadotropin", J. Clin Endocrinol Metab. (1990) 70:783-87) . Pregnancy serum β-core fragment levels have been reported to be extremely low, 0.02 -0.3% of hCG level (Wehmann et al supra and Alfthan et al, supra) . This has led to a conclusion that serum levels cannot account for the high levels found in urine, and that β-core fragment must therefore be the result of renal degradation of circulating hCG or the β-subunit. As such, there was controversy as to whether the β-core fragment is a secretory product of the cells versus a degradation product by the kidney of circulating hCG or its β-subunit. Two recent studies show that β-core fragment levels in urine are highest at 12-16 weeks of pregnancy, when serum hCG and β-subunit levels are continuously declining (Cole et al, Mol. Endocrinol, 1988; 2:825-30 and Kato, Y., Braunstein, G.D., "β-Core Fragment is a Major Form of Immunoreactive Urinary Chorionic Gonadotropin in Human SUBSTITUTESHEET Pregnancy", J. Clin Endocrinol Metab. (1988) 66:1197-1201). This showed discordance between the levels of these molecules, and that serum β-subunit/HCG may not be the source of urine β-fragment. By immunohistochemistry, the β-core fragment has been detected in trophoblast tissue and in 93% (77/83) of an assortment of tumor sections (Kardana et al, Br. J. Cancer, 1988, 58:281-6). Forms of β-core fragment, have also been detected in cultures of the DoT and CaSki cervical cancer cell lines (Hussa, R.O., Fein, H.G. , Pattillo, R.A. , Nagellberg, S.B., Rosen, S.W., Weintraub, B.D., Perini, F. , Ruddon, R.W. , Cole, L.A. , "A distinctive Form of Human Chorionic Gonadotropin β-subunit-like Material Produced by Cervical Carcinoma Cells", Cancer Res. (1986) 46:1948-54). Furthermore, high levels of the β-core fragment have been demonstrated in the fluids of 24 h trophoblast organ cultures (Cole et al, Mol. Endocrinol, 1988; 2:825-30). Studies have shown that urinary β-core fragment injected into humans has a very rapid metabolic clearance rate (Wehmann, R.E., Blithe, D.L., Flack, M.R., Nisula, B.C., "Metabolic Clearance Rate and Urinary Clearance of Purified β-core", J. Clin Endocrinol Metab. (1989) 69:510- 17) . These studies, however, had two major limitations. Firstly, they recovered only 8% of the injected material. Secondly, purified urinary β-core fragment may have already been processed for excretion. A more accurate assessment of clearance rate would be obtained by measuring the postpartum disappearance of serum β-core fragment complex, which represents the major form found in serum. Copending U.S. patent application Serial No. 07/204,447, filed June 8, 1988 describes methods for detecting the onset, progression and regression of gynecologic cancers by assaying a urine sample for hCG β- subunit or β-core fragment. Cervical cancer is one of the most common malignancies afflicting women (E. Silverberg, "Cancer Statistics", CA-A Cancer J. Clinicians. 6, 9-26, (1986)). The Pap (Papanicolaou) smear has led to early diagnosis and has mainly been responsible for the overall improvement in survival reported for this disease (Yajima, A., Mori, T. , Sato, S., Wakisaka, T., and Suzuki, M. , "Effect of Cytologic Screening on the Detection of Cervical Carcinoma", Obstet. Gynecol. , 59., 565-568 (1982)). However, patients who are not able or do not wish to undergo a Pap smear screening run the risk of unsuccessful treatment if they wait for symptoms (pain, bleeding and/or discharge) to develop. This often is the situation with women who are in epidemiologic groups at highest risk for developing cervical cancer. For example, the National Cancer Institute of Peru recently reported that 80% of 1,100 new cervix cancer patients seen annually have stage III or IV disease. For patients who do not submit to routine Pap Smear screening, the survival is extremely poor (Castellano, C. , "Manejo Del Paciente Con Citologia Anormal", Ninth Congress, Cancer in Peru, Lima, (1985)). The Pap smear as a screening technique for cervical cancers can also be inaccurate. False negative rates for Pap smears vary greatly from 12.5% to 45% (C. Castellano, supra; Jordan, S. W. , Smith N. L. , and Dike, L. S., "The Significance of Cervical Cytologic Dysplasia", Acta Cytol. , 25, 237-244, (1981)). Among American women, the ovary is the second most common site of gynecologic cancer (E. Silverberg, supra) . The common epithelial ovarian cancers lack early warning symptoms and there are no routine tests, like the Pap smear, SUBSTITUTESHEET for early detection. Ovarian cancer is usually not suspected until a pelvic mass is present and, if not detected until advanced stage, is almost always fatal (Schwartz, P. E. , "Gynecologic Cancer", In: J. A. Spittle, Jr. (ed.), Clinical Medicine, pp. 1-44. Philadelphia: Harper and Row, (1985)). A readily available blood test that would aid in the detection of women at increased risk for having gynecologic malignancies, particularly cervix and ovarian cancer would be a major step forward for patients in whom advanced stage disease is almost always fatal. Such a test may obviate the need for pelvic examination and Pap smears. Furthermore, blood tests do not require the need for a trained physician as required for pelvic examinations and Pap smears. The efficacy of treatment for patients with recurrent gynecologic cancer is reflected in the volume of cancer at the time recurrence is documented and the sites of recurrent disease. Therapy may be of limited value when recurrent disease is not identified until the patient has clinical signs or symptoms. Early recognition of persistent or recurrent cancer may lead to more effective therapeutic intervention. Surgical intervention in the form of radical surgery may cure patients with central recurrence of cervical cancer. Diagnosis of persistent or recurrent central disease in a radiation field may be difficult to confirm by cytologic or biopsy techniques. An accurate tumor marker for cervical cancer may lead to earlier recognition and a more rapid diagnosis and treatment. Previous experience suggested a role for lipid-associated sialic acid (LASA) in this regard, but not for squamous cell carcinoma antigen (SCC) (Schwartz, P.E., SUBSTITUTESHEET Foemmel, R.S., Chambers, S.K., and Chambers, J.T., "Evaluation of Squamous Cell Carcinoma Antigen (SCC) and Lipid-association Sialic acid (LSA) in Monitoring Patients With Cervical Cancer", Proc. Am. Soc. Clin. Oncol.. (5, 113 (1987) ) . Early recognition of recurrent or persistent endometrial cancer may lead to more rapid treatment with potentially more effective combination chemotherapy (Seski, J.C., Kasper, G.L., and Kunschner, A. . , "Chemotherapy for Endometrial Cancer, in Diagnosis and Treatment Strategies" (F.N. Ru ledge, R.S. Freedman and D.M. Gershenson Eds.), University of Texas Press, Austin, pp. 327-334 (1987)). Multiple circulating markers have been evaluated in the management of epithelial ovarian cancer patients, the most promising of which is CA 125 (Bast, R.C., Kung, T.L., St. John, E., Jenison, E., Niloff, J.M., Lazarus, H. , Berkowitz, R. , Leavitt, T. , Griffiths, T., Parker, L. , Zurawski, V.R. , and Knapp, R.C., "A Radioimmunoassay Using a Monoclonal Antibody to Monitor the Course of Epithelial Ovarian Cancer", N. Engl. J. Med.. 309, 883-887 (1983)). As high as eighty percent of patients with non-mucinous ovarian cancers can have elevated levels of CA 125 in their serum, which will become nondetectable as the cancer responds to treatment. Unfortunately, in a recently reported clinical trial using CA 125 in the management of ovarian cancer, 6 of 11 (55%) patients who were clinically free of disease and had CA 125 levels that were in the normal range, were found to have persistent cancer at a second-look procedure (Atack, D.B., Nisker, J.A. , Allen, H.H. , Tustanoff, E.R., and Levin, L. , "CA 125 Surveillance and Second-look Laparotomy in Ovarian Carcinoma", Am. J. Obstet. Gvnecol.. 154. 287-289 (1986)). SUBSTITUTESHEET Similarly, at Yale-New Haven Hospital the published false-negative rate for CA 125 (cut-off 35 U/ml) at second-look surgery is 40% (Schwartz, P.E., Chambers, S.K., Chambers, J.T., Gutmann, J. , Katopodis, N. , and Foemmel, R.S., "Circulating Tumor Markers in the Monitoring of Gynecologic Malignancies", Cancer. 60. 353-361 (1987)). Other early clinical trials have shown that decreasing CA 125 levels in ovarian cancer patients are not necessarily an indicator of regressive disease (Alvarez, R.D., To, A., Boots, L.R., Shingleton, H.M. , Hatch, K.D., Hubbard, J., Soong, S.J., and Potter, M.E., "CA 125 as a Serum Marker for Poor Prognosis in Ovarian Malignancies", Gynecol. Oncol. f 16, 284-309 (1987)). Once the diagnosis of ovarian cancer is established, the currently available clinical markers tend to parallel clinical examination findings, but do not guarantee the efficacy of the treatment in those patients who are clinically free of disease, nor are they sufficiently sensitive to avoid the use of second-look operations. Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of the following two dissimilar subunits: alpha 92 amino acids long and beta 145 amino acids long, joined non-covalently. It is normally produced by trophoblast tissue and can be detected in the blood and urine of women in pregnancy or trophoblast disease. Free forms of hCG alpha and beta subunits, which can account for 90% of the total produced, are also found in blood and urine in pregnancy and trophoblast disease (Cole, L. A., Kroll, T. G. , Ruddon, R. W. , and Hussa, R.O., "Differential Occurrence of Free B and Free A Subunits of Human Chorionic Gonadotropin in Pregnancy Sera", J. Clin. Endocrinol. Metab. , 58, 1200-1202 (1984); Cole, L.A. , "Occurrence and Properties of Glycoprotein Hormone Free Subunits, in SUBSTITUTE SHEE Microheterogeneitv of Glycoprotein Hormones (H. Grotjan and B. Keel, Eds.), CRC Press, New York; Cole, L.A. , Hartle, R. . , Laferla J.J. , and Ruddon, R.W. , "Detection of the Free beta-subunit of Human Chorionic "Detection of the Free beta-subunit of Human Chorionic Gonadotropin In Cultures of Normal and Malignant Trophoblast Cells, Pregnancy Sera, and Sera of Patients with Choriocarcinoma", Endocrinology, 113, 1176-1178 (1983)). Numerous researchers have shown that human chorionic gonadotropin is present in the circulation of approximately 20% of women with cancer. Because of the low percentage positive for cancer it has not been used as a marker for gynecological cancer. Free beta-subunit, asialo free beta and the core fragment of asialo beta-subunit, together called "UGF", are rapidly cleared from the circulation and are more-readily detected in urine than in serum samples (Schroeder, H.R. , and Halter, CM., "Specificity of Human beta- Choriogonadotropin Assays for the Hormone and for an Immunoreactive Fragment Present in Urine During Normal Pregnancy", Clin. Chem.. 29, 667-671 (1983) ; Lefort, G.P., Stolk, J.M., and Nisula, B.C., "Renal Metobolism of the beta-subunit of Human Choriogonadotropin in the Rat", Endocrinology. 119. 924-931 (1986); Wehmann, R.E., and Nisula, B.C., "Metobolic Clearance Rates of the Subunits of Human Chorionic Gonadotropin in Man", J. Clin. Endocrinol. Metab.. 48., 753-759 (1979)). Ectopic hCG has been detected in the blood and tissues of patients with non-trophoblastic cancers, most notably gynecologic malignancies (R.O. Hussa, "Human Chorionic Gonadotropin, a Clinical Marker: Review of its Biosynthesis", Ligand Rev.. 3. . 1-43, (1981)). SUBSTITUTE SHEET In a recent compilation of 38 separate studies (n = 692) , 36% of women with cervical, 27% of those with endometrial and 13% of those with vulvar cancers were shown to have detectable levels of hCG in radioimmunoassays. This low percentage with detectable levels and the associated low titers have, however, restricted the use of hCG in detecting and following the therapy of gynecologic cancers. In studies of women with gynecologic cancer, using the Hybritech "Tandem" hCG-specific immunoradiometric assay (<0.1% hLH and free subunit crossreactivity) , it was found that only 11 of 64 (17%) had detectable (>0.2 ng/ml, equivalent of >2.0 mlU/ml) serum levels of hCG (Wang, Y., Schwartz, P.E., Cole, L.A, "Serum hCG Investigation in Patients with Non-trophoblastic Cancer", J. Obstet. Gvnecol. China, in press) . The average level was found to be just 0.30 ng/ml (equivalent of 3 mlU/ml) . Serial serum samples from 14 cancer patients with elevated hCG levels were examined. Levels were measured at the start and following therapy. Strangely, in 8 of 10 women with progressive cancer and increasing tumor mass hCG levels went down (Wang et al, supra) . Furthermore, in 4 of 4 women with regressing disease and diminution of tumor mass, hCG levels went up. Clearly, serum hCG has very limited value in screening and in the management of patients with gynecologic cancer. The hCG free beta-subunit and core fragment, UGF, have also been detected in patients with non-trophoblastic cancers (Papapetrou, P.D., and Nicopoulou, S.C., "The Origin of a Human Chorionic Gonadotropin beta-subunit Core Fragment in the Urine of Patients with Cancer", Endocrinologica, 112 , 415-422 (1986); Vaitukaitis, J.L., "Characterization of a Small Molecular Size Urinary Immunoreactive Human Chorionic Gonadotropin (hCG)-like Substance Produced by Normal Placenta and by hCG-secreting Neoplasms", J. Clin. Endocrinol. Metab.. 53., 1014-20 (1981)). SUBSTITUTESHEET In patients with UGF in urine, the fact that it originates from the cancer tissue itself has been established by the finding of significant levels (average 1.0 ng/mg protein) in 5 of 5 tumor tissue ho ogenates. In a preliminary study of UGF in patient urines (Cole, L.A., Wang, Y., Elliott, M. , Latif, M. , Chambers, J.T., Chambers, S.K., and Schwartz, P.E., "Urinary Human Chorionic Gonadotropin Free beta-Subunit and beta-Core Fragment: A New Marker of Gynecologic Cancers", Cancer Res. , 48, 1356-1360, (1988)) levels were measured in spot samples from 50 healthy women and from 68 patients with active gynecologic cancer. Elevated levels (>θ.2 ng/ml beta-subunit, or molar equivalent of the beta core fragment) were detected in 3 of the control and 50 of the cancer samples. Although adjustments were not made for urine concentration (creatinine level) , a sensitivity of 74 % and a specificity of 94% of UGF for gynecologic cancers was suggested. These preliminary findings showed that gynecologic cancers more commonly produce free-subunits or beta core fragment, than hCG, and that the use of UGF as a tumor marker warranted further investigation. DEFINITIONS hCG - human chorionic gonadotropin Tumor marker - a molecule that specifically identifies a specific tumor "SGPC" or "SGP complex" or "serum gonadotropin peptide complex" - a complex of one or two molecules, namely β-core fragment (intracellular post-translatonal processing product of pre-β-subunit of hCG, which cannot be made from the complete β-subunit of hCG) or onomeric β-core fragment (same origin as the β-core fragment) with a carrier protein; SUBSTITUTE SHEET the molecules are present in differing quantities in culture fluids in an assortment of cancer cell lines; the carrier protein masks the epitopes of these two molecules so that they cannot be detected by conventional methods, i.e, the molecules cannot be detected by current immunoassays for (1) hCG, for (2) hCG beta subunit (also called "beta subunit") , for (3) hCG beta subunit core fragment (also called "beta core fragment") or for (4) hCG beta subunit C terminal peptide or other hCG antigens, by presently existing methods; the physical characteristics of these two molecules (such physical characteristics being based on dissociated molecules) indicate that the β-core fragment is similar to hCG beta subunit residues 6 to 40 disulfide linked to hCG beta subunit residues 55 to 92 and having a distinctly different oligosaccharide structure compared to hCG or its beta subunit; the monomeric β-core fragment appears to be similar to and possibly represents beta subunit residues 1 to 92 or 6 to 92 and is a monomer with different oligosaccharide structure compared to hCG or its β-subunit; the complex as present in serum has a molecular size of approximately 70,000 daltons, or slightly larger than hCG. "SGP" or "serum gonadotropin peptides" - the molecules that comprise the portion of SGPC that does not contain the carrier protein (described hereinbefore as the "molecules") ; the unmasked portion of SGPC that is sought to be analyzed; there are believed to be two peptides which comprise SGP, namely (1) β-core fragment, a peptide of amino acids 6 to 40 bonded by disulfide bridges to a peptide of amino acids 55 to 92 of the 1 to 145 amino acid sequence of human chorionic gonadotropin beta-subunit and (2) monomeric β-core fragment, a peptide of amino acids 6 to 92 or 1 to 92 of the 1 to 145 amino acid sequence of human chorionic gonadotropin beta- subunit, each having different attached oligosaccharides to hCG or its β-subunit. SUBSTITUTESHEET HT jGF" - the free beta-subunit, asialo free beta and the core fragment of asialo beta subunit are together called UGF. SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for detecting or following the course of a cancer, e.g., a non-trophoblastic cancer, by analyzing blood or a fluid thereof. It is a further object of the present invention to provide a method for detecting or following the course of pregnancy or a trophoblastic disease by analyzing blood or a fluid thereof. It is still another object of the present invention to break up the serum gonadotropin peptide complex to expose one or more epitopes thereof in serum gonadotropin peptides. These objects as well as other objects, aims and advantages are satisfied by the present invention. Applicants have discovered that the regular and monomeric beta core fragments which together are one part of what is herein called "serum gonadotropin peptide complex" are in fact present at high levels in blood/serum/plasma, but are bound tightly to a much larger, unrelated, molecule, in such a way that they loose all recognition by hCG, hCG β- subunit and β-core fragment tests. Applicants have discovered methods to break up this complex, expose it and thus quantitate this important molecule. The present invention concerns a method for detecting and following the course of a cancer, e.g., a non- trophoblastic cancer, comprising analyzing whole blood or a SUBSTITUTESHEET fluid of whole blood for the presence of serum gonadotropin peptide complex or a component of serum gonadotropin peptide complex, wherein the presence of serum gonadotropin peptide complex or a component thereof is an indication of the presence of a cancer, e.g., a non-trophoblastic cancer, pregnancy or a trophoblastic disease in a patient from which the blood or fluid of whole blood was obtained. The present invention is also directed to a method of breaking up serum gonadotropin peptide complex to expose one or more epitopes thereof comprising contacting serum gonadotropin peptide complex or a fluid containing the same with a dissociation agent or by dissociation by heating at a temperature above 60"C. More particularly, this aspect of the invention relates to contacting blood, serum or plasma with a dissociation agent, i.e, a detergent, a chaotropic agent, an acid having a pH of 2 to 3 or an organic acid, or by heating the blood, serum as plasma to a temperature above and then conducting a separation to remove the dissociation agent or to separate out the serum gonadotropin peptide complex or a component thereof. The present invention is further directed to a composition comprising a serum gonadotropin peptide free from serum gonadotropin peptide complex in blood or a fluid thereof, e.g., plasma or serum. The present invention further relates to a substantially pure (or purified) serum gonadotropin peptide complex comprising at least one β-core fragment selected from the group consisting of a regular β-core fragment and a monomeric β-core fragment, in association with a higher molecular weight carrier molecule which immunologically masks at least a substantial portion of the contained β-core fragments, said complex being further characterized by: (a) a molecular weight about 70,000 in serum as measured by gel filtration and (b) dissociation to release at least one β-core fragment having a molecular weight of about 15,000 daltons upon treatment with 3M ammonium thiocyanate. BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1A to IE are graphs which represent the results for gel filtration of serum. Figs. IF to IJ are graphs which represent gel filtration of serum fractions (according to an embodiment of the present invention) . In Figs. 1A to IJ, results are depicted for fractions tested for hCG ( ) , free β-subunit fragment (. .) and β-core fragment (. .) by immunoassays. Figs. 1A, IB, 1C and ID depict results for early, mid-and term pregnancy, and choriocarcinoma serum, respectively. Figs. IF to II are repeat chromatography of the POOLS marked A, B, C and D (after dissociation with ammonium thiocyanate, NH 4 SCN) . Fig. IE depicts the results for pregnancy serum, untreated and Fig. IJ depicts the results for pregnancy serum directly treated with NH 4 SCN (right) . Elution positions of urinary hCG, β-subunit and β-core fragment standards are indicated by arrows, and of blue dextran (V 0 ) , ovalbumin (mw=43,000) and myoglobin (mw=17,000) by the symbols φ, (§ and Q), respectively. SUBSTITUTESHEET DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the finding that the regular and monomeric β-core fragments in serum is associated with other molecules, to form a high molecular weight complex, which denies recognition by existing antibodies to the β-subunit and β-core fragment epitopes. This complex can be dissociated with, for example, 3M ammonium thiocyanate to release the β-core fragment. Levels of released β-core fragment (relative to hCG) are comparable to those reported for tissue and urine. The level of "free" β-core fragment in serum, as conventionally detected by immunoassay/gel filtration is extremely low (beyond detection by conventional immunoassays) . However, applicants discovered that the major species of β-core fragment is a component of a large molecular weight complex (Mr>60,000), that is immunologically masked and can be dissociated to produce a molecule that elutes on gel filtration (Mr = 15,000) and on reverse-phase HPLC in the position of urine β-core fragment standard. The proportion of β-core fragment found in early pregnancy serum was 18% of the hCG level, that in mid- pregnancy was 91% and in term pregnancy 50% of the hCG level. By comparison, urine β-core fragment levels are 35%, 490% and 250% of hCG levels, respectively, for equivalent stages of pregnancy. Applicants discovered the presence of a masked form of β-core fragment in pregnancy serum with levels that rise and fall concordant with urine β-core fragment values (lowest β- core fragment level in early pregnancy, highest at 13-16 weeks) . It is considered that this is a likely source of urine β-core fragment. Levels of masked β-core fragment (untreated SGPC) in serum are approximately one quarter β- core fragment levels in urine. Considering that serum creatinine levels are 1/20-1/40th of urine levels, such levels of masked β-core fragment in serum are more than ample to account for urine levels. The finding of significant levels of forms of β-core fragment in tissue, serum and urine, indicates a native cellular origin for this molecule. Recently, Wehmann and colleagues (Wehmann et al, J. Clin., Endocrinol. Metab.. 1990, 70; 371-78) added urinary β-core fragment to normal serum, and found its molecular size (by gel filtration) remained unchanged. They concluded that β-core fragment immunoreactivity was not obscured by serum factors. While it is realized that the urinary β-core fragment used by Wehmann and colleagues may not be the same as that which forms the serum complex, these findings do suggest that the serum complex is not a result of non¬ specific binding of the β-core fragment to random serum proteins; the β-core fragment is not only present in serum as a complex, but has been consistently detected in urine associated with two small unrelated peptides (Kardana et al., submitted for publication). These small peptides have a minor effect on the molecular size of urinary β-core fragment, and can be separated by reverse phase HPLC. It is considered that perhaps the urine β-core-peptide complex is the product of processing of the masked serum β-core fragment material. It is clear that the β-core fragment story is not a simple one. While not eliminating a urinary degradation pathway, that may co-exist, the finding of high levels of β- core fragment in the serum gives weight to a secretory origin. T One embodiment of the present invention involves the breaking up of serum gonadotropin peptide complex to expose one or more epitopes thereof. This is accomplished by exposing SGPC for a sufficient period of time to a dissociation agent. Thus, blood, plasma or serum, for example, is contacted with a dissociation agent such as a detergent, e.g., sodium lauryl sulfate, "TRITON X100" ("TRITON" is a surfactant based on alkylaryl polyether alcohols, sulfonates and sulfates; nonionic, cationic and anionic types, oil-soluble and water-soluble types) or NP40 (octylphenylethylene oxide) ; a chaotropic agent, e.g. guanidine, ammonium thiocyanate or urea; an acid having a pH of 2 to 3, e.g., citric acid, formic acid or acetic acid or an organic solvent, e.g., toluene, acetonitrile, acetone or methanol. The dissociating agent is any agent which serves to dissociate non-covalently linked proteins. Alternatively, blood, plasma or serum is heated to greater than 60°C. The treated blood, plasma or serum is then subjected to a separation step to remove precipitates and/or separate out the SGPC or component thereof or to separate out the dissociation agent. Techniques for separation are well known in the art and, e.g., a hydrophobic-interaction column, gel filtration, reverse phase high pressure liquid chromatography, the use of membranes, osmosis, electrophoresis, dialysis, hydroxyapatite columns, ion exchange or σentrifugation can be utilized. Once dissociated from the SGPC, the SGP can be detected by any convenient hCG β-subunit or β-core fragment assay, for example, ELISA, RIA, dyes, fluorometric assays or EIMA (enzyme immunometric assay) . T TESHEET The SGPC and SGP can also be detected in a non- dissociated state by the use of polyclonal antibodies or monoclonal antibodies, said antibodies specific to SGPC. The two steps of dissociation and separation may be combined in a single operation by using, for example, reverse phase high pressure liquid chromatography (HPLC) , wherein the solvent utilized therein may act as the dissociation agent. The following of the course of a cancer, pregnancy or a trophoblastic disease according to the present invention involves the detecting of the progression or regression of the non-trophoblastic cancer or trophoblastic disease or the progression of a pregnancy comprising (a) taking a first measurement of SGPC, e.g., by using an antibody, or a component thereof, e.g., SGP, e.g., after dissociation of SGPC or by using an antibody, in blood or a fluid component thereof, e.g., serum or plasma, from a patient; (b) subsequent to step (a) , taking one or more further measurements of SGPC or a component thereof, e.g., SGP, in blood or a fluid component thereof, e.g., serum or plasma from said patient, and (c) comparing the first measurement from (a) to the measurement(s) from (b) to ascertain if SGPC or SGP is increasing or decreasing, as an indication of progression of the pregnancy, a cancer or trophoblastic disease (if there is an increase) , or regression of the cancer or trophoblastic disease (if there is a decrease) . SUBSTITUTESHEET The invention also encompasses kits comprising in one or more containers, means to detect SGP or SGPC, e.g., an antibody, in blood or a fluid thereof and optionally means to dissociate SGPC, e.g., a dissociation agent as described herein. Non-limiting examples of uses for the present invention are as follows: 1. IVF (in vitro fertilization) Programs: hCG is given during the IVF procedure. Administered hCG has to completely leave the circulation system (takes 2-3 weeks) before endogenous hCG can be detected and pregnancy demonstrated. With the development of sensitive methods, detection of a separate molecule beta core fragment - carrier complex, could establish pregnancy after just 7-10 days. 2. hCG is useful for monitoring only the first 8-10 weeks of pregnancy, after this time levels indiscriminately fall. Levels of SGP and SGPC do not fall until approximately 16 weeks of pregnancy and thus have an advantage over hCG in monitoring pregnancy/placental health. 3. Urine measurement of beta core fragment (the urine form of SGP) have proven to be useful in the detection and management of ovarian, uterine and endometrial cancers. There has, however, been some reluctance to break away from the traditional serum/blood measurement of tumor markers and start examining urines. Further reluctance has come from the 2-3-fold variation in urine concentrations at different times of the day. The discovery of SGPC and means to expose epitopes thereon provides a means of measuring this useful marker in the blood/serum. SUBSTITUTESHEET 4. Detection and following the course of cancers, e.g., ovarian cancer, cervical cancer, endometrial cancer, breast cancer, vaginal cancer, vulvar cancer, lung cancer, colon cancer, bladder cancer and pancreatic cancer. 5. Detection of pregnancy disorders, e.g., threatened abortion, ectopic pregnancy and toxemia. 6. Most tumor markers presently available are for blood, not urine, and panels containing several of such markers are generally employed. SGP could be one of such markers on such panel. Detection of tumor markers in blood has been found to be more reliable than in urine, since urine sampling is affected, for example, by an individual's intake of fluid prior to testing. EXAMPLES The present invention is now described with reference to the following non-limiting examples. Example 1 Four pools of serum were used for β-core fragment investigations: (i) 6 early pregnancy serum samples (5th week after menstrual period) ; (ii) 2 mid-pregnancy serum samples (13 and 16 weeks) ; (iii) 2 term pregnancy serum samples; (iv) 4 serum samples from women prior to therapy for choriocarcmoma. An aliquot (3-4ml) of each pool was gel filtered on a double-column of Sephacryl S-100 HR (two 1.6 x 90cm columns, connected in series) equilibrated in 0.1M ammonium bicarbonate. The column was pumped at 30ml/hr and 2ml fractions collected. High molecular weight fractions (Mr>60,000) containing small amounts of β-core fragment SUBSTITUTE SHEET immunoreactivity (<0.1% of hCG level) were pooled (Fig. 1), lyophilized and reconstituted in 4ml 3M ammonium thiocyanate. After 15 minutes at room temperature, samples were centrifuged and re-applied to the S-100 HR column. To avoid instant reassociation, 3ml of 3M ammonium thiocyanate was put on the column prior to sample application. Alternatively, solid ammonium thiocyanate was added directly to the serum pool (to 3M) , left 15 minutes at room temperature, centrifuged then applied to the S-100 HR column. Controls consisting of hCG standard (batch CR-127, from NIH) in 3M ammonium thiocyanate, hCG standard in normal serum containing 3M ammonium thiocyanate and β-subunit standard in 3M ammonium thiocyanate, were also left at room temperature for 15 minutes and then put on the S-100 HR column. To eliminate interference from trace amounts of molecules non-specifically bound to the gel (β-core fragment and proteins used to calibrate column) , the column was routinely stripped, prior to use, with 8 ml 3M ammonium thiocyanate. Eluent was then checked with immunoassays and shown to be free of β-core fragment. Immunoassays: HCG dimer was measured using a modification of the Tandem-E kit from Hybritech (San Diego, CA) . This assay, which requires the presence of an α and a β-subunit, has 0% (wt/wt) cross-reactivity with free β- subunit and β-core fragment. Free β-subunit was measured in an assay using monoclonal 1E5 coated onto microtitre plates (Hybritech) . This assay has 3% cross-reactivity with hCG and 0% with β-core fragment, β-core fragment was measured using monoclonal B204 (Columbia College of Physicians and Surgeons) coated onto microtitre plates. This assay has 0.6% cross-reactivity with hCG and 9% cross-reactivity with free β-subunit (Cole et al, Cancer Res. , (1983) 48:1356-60; SUBSTITUTESHEET O'Connor et al, Cancer Res. , 1988:48:1361-66; Alfthan et al, J. Clin Endocrinol. Metab. (1990) 70:783-87). All three immunoassays utilized a detector step consisting of β- subunit polyclonal antibody linked to horseradish peroxidase (Bios Pacific Inc. , CA) . Results: Gel filtration of pools of early, mid- and term pregnancy serum, and of choriocarcinoma serum are shown in Figures 1A-D. As shown, immunoreactivity in the position of the β-core fragment standard (Mr = 15,000) never exceeded 0.1 mol/1 or 0.1% of the hCG level. Repeat chromatography of higher molecular weight fractions (Mr>60,000) dissociated with ammonium thiocyanate, however, generated vastly greater amounts of immunoreactivity in the β-core fragment region (Mr = 15,000). After dissociation, 19 pmol (pica moles) of β-core fragment was generated from early pregnancy serum (18% of hCG level) , 311 pmol from mid-pregnancy serum (91% of hCG level) , and 58 pmol from term pregnancy serum (50% of hCG level) . Choriocarcinoma serum, however, released just 5.4 pmol (1.3% of the hCG level). Reverse phase HPLC using the method in Birken et al, Endocrinology, (1988), 123; 572- 83 was used to compare the molecule released from mid- pregnancy serum with urine β-core fragment standard. Both molecules again eluted in the same position, confirming the identity of the released molecule as β-core fragment. When early pregnancy serum was directly treated with ammonium thiocyanate (Fig. IJ) , no peak was observed in the position of the β-core fragment (Mr = 15,000). Instead, several small peaks with β-core fragment immunoreactivity were detected (Mr 20-60,000). Without wishing to be bound to any particular theory of operability, it is believed that perhaps the additional mass of serum proteins interfere in SUBSTITUTE SHEET the separation, or reassociate with β-core fragment to form intermediate complexes. In control experiments, no β-core fragment was generated from the incubation of hCG or β-subunit standards with ammonium thiocyanate. This demonstrates that the findings are not due to dissociation or damage of hCG or its β-subunit by the ammonium thiocyanate protocol. To summarize the above results, in pools of serum from several individuals (human females) with first trimester pregnancies, SGPC was detected at a level of approximately 20% of hCG. In second trimester pooled serum, SGPC was found at levels similar or equal to hCG (urine β-core fragment levels are highest in the second trimester of pregnancy) . In third trimester pooled serum, levels of SGPC were approximately half of that of hCG. Accordingly, of the three trimesters, higher levels of SGPC were seen in the second trimester. Without wishing to be bound by any particular theory of operability, this suggests a relationship between serum levels and urine levels of the beta core fragment. Example 2 Serum was taken from three women with ovarian cancer, including one woman who was (falsely) negative for the beta core fragment (human chorionic gonadotropin beta-subunit core fragment) in urine and who had a markedly elevated level of CA 125 in serum, consistent with advanced disease and clinical manifestations of advanced disease. All three women, including the "false negative" woman, had relatively high serum levels (2.5 to 32.1 picomoles/ml or 2.5 to 32.1 x 10 "12 moles/ml) of SGPC versus 3 to 100 femtomoles per ml or 3 to 100 x 10 '15 moles/ml of beta core fragment in urine. SUBSTITUTESHEET In a large pool of normal serum from healthy, non- pregnant women, the serum level of the beta core fragment was found to be 0.25 picomoles/ml or 0.25 x 10 "12 moles/ml. In human females which received two injections of "PREGNYL" (a crude hCG preparation) , a similar level of SGPC was seen (0.3 picomoles/ml or 0.3 x 10 "12 ) . This finding was consistent with SGPC being associated with an endogenous source of two molecules. Examination of a pool of serum from pregnant women directly after a missed menstrual period revealed a SGPC level of 12.1 picomoles/ml or 12.1 x 10 "12 moles/ml or almost 50 times the background. One week after ovulation or after an in vivo fertilization procedure SGPC levels would be approximately 0.5 picomoles/ml or 0.5 x 10 "12 moles/ml, or positive for pregnancy. The following culture fluids of two trophoblastic cancer cell lines were examined: JAR (trophoblastic) cell line secreted hCG and SGPC; OVCA (ovarian cancer) cell line secreted only SGPC. Without wishing to be bound by any particular theory of operability, this suggests that SGPC is a direct secretion product of cancer cells and that SGPC is secreted as such and that individuals components to not form SGPC in vivo. Results for Example 2 are summarized below in Table 1 SUBSTITUTESHEET TABLE 1 Values in Table 1 are in picomoles/ml (10 *12 moles/ml) starting serum It will be appreciated that the instant specification is set forth by way of illustration and not limitation, and that various modifications and changes may be made without departing from the spirit and scope of the present invention. SUBSTITUTESHEET

Claims: IN THE CLAIMS : 1. A method for detecting or following the course of a cancer comprising analyzing whole blood or a fluid of whole blood for the presence of serum gonadotropin peptide complex or a component of serum gonadotropin peptide complex, wherein the presence of serum gonadotropin peptide complex or a component thereof is an indication of the presence of a cancer in a patient from which said blood or said fluid of whole blood was obtained. 2. The method of claim 1, wherein said fluid of whole blood is plasma. 3. The method of claim 1, wherein said fluid of whole blood is serum. 4.- The method of claim 1, wherein said cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, breast cancer, vaginal cancer, vulvar cancer, lung cancer, colon cancer, bladder cancer and pancreatic cancer. 5. The method of claim 1 wherein the component of serum gonadotropin peptide complex is a serum gonadotropin peptide. 6. The method of claim 1, wherein the component of serum gonadotropin peptide complex is a carrier protein. 7. The method of claim 1, wherein the whole blood or fluid thereof is contacted with a dissociation agent or heated to achieve a dissociation, and a separation is conducted to separate out said serum gonadotropin peptide complex or a component thereof. SUBSTITUTESHEEI 8. The method of claim 7, wherein said dissociation agent is selected from the group consisting of a detergent, a chaotropic agent, an acid having a pH of 2 to 3 and an organic solvent. 9. The method of claim 8, wherein said dissociation agent is selected from the group consisting of sodium dodecyl sulfate, octylphenylethylene oxide, guanidine, urea, ammonnium thiocyanate, acetic acid, citric acid, formic acid, toluene, acetonitrile, methanol and acetone. 10. The method of claim 7, wherein said separation is conducted by hydrophobic-interaction column, ion exchange, gel filtration, reverse phase high pressure liquid chromatography, dialysis, ultrafiltration, osmosis, electrophoresis, hydroxyapatite column or centrifugation. 11. The method of claim 1, wherein said complex or a component is detected by an antibody specific to said complex or to said component thereof. 12. The method of claim 1, wherein said antibody is a polyclonal antibody. 13. The method of claim 11, wherein said antibody is a monoclonal antibody. 14. A method of detecting or following the course of pregnancy or a trophoblastic disease comprising analyzing whole blood or a fluid of whole blood for the presence of serum gonadotropin peptide complex or a component of serum gonadotropin peptide complex, wherein the presence of serum gonadotropin peptide complex or a component thereof is an indication of the presence of pregnancy or a trophoblastic SUBSTITUTESHEET disease in a patient from which said blood or said fluid of whole blood was obtained. 15. The method of claim 14, wherein said fluid of whole blood is plasma. 16. The method of claim 14, wherein said fluid of whole blood is serum. 17. The method of claim 14, wherein the component of serum gonadotropin peptide complex is a serum gonadotropin peptide. 18. The method of claim 14, wherein the component of serum gonadotropin peptide complex is a carrier protein. 19. The method of claim 14, wherein the whole blood or fluid thereof is contacted with a dissociation agent or heated to achieve dissociation, and a separation is conducted to separate out said serum gonadotropin peptide complex or a component thereof. 20. The method of claim 19, wherein said dissociation agent is selected from the group consisting of a detergent, a chaotropic agent, an acid having a pH of 2 to 3 and an organic solvent. 21. The method of claim 20, wherein said dissociation agent is selected from the group consisting of sodium dodecyl sulfate, octylphenyl ethylene oxide guanidine, urea, ammonium thiocyanate, acetic acid, citric acid, formic acid, toluene, acetonitrile, methanol and acetone. SUBSTITUTE SHEET

Inventors: COLE LAURENCE A, KARDANA ANDREW, COLE, LAURENCE, A., KARDANA, ANDREW
Applicants: COLE LAURENCE A, KARDANA ANDREW, UNIV YALE, COLE, LAURENCE, A., KARDANA, ANDREW, YALE UNIVERSITY
Application year: 1991
Main classification: C07K15
IPC codes: G01N33