CA2264277C - Methods and kits for obtaining and assaying mammary fluid samples for breast diseases, including cancer - Google Patents

Abstract

Non-invasive methods and kits are provided for obtaining biological samples of mammary fluid or mammary fluid components by administering oxytocin or an oxytocin analogue to a mammalian patient to stimulate expression of mammary fluid. The oxytocin is preferably administered intranasally and causes myoepithelial contraction of target alveolar-ductal tissues of the breast. During or after mammary fluid expression, a biological sample is collected in the form of whole mammary fluid, whole cells or cellular components, other selected liquid or solid fractions of the mammary fluid, purified or bulk proteins, glycoproteins, peptides, nucleotides or other desired constituents of mammary fluid. Methods and kits are also provided for determining the presence or amount of a breast disease marker in biological samples of mammary fluid or mammary fluid components. Samples obtained according to the above sample collection methods are assayed to determine the presence and/or amount of a breast disease marker, for example a breast cancer marker such as CEA, HMFG, MCA, vasopressin, or cathepsin D, in the sample. Cellular samples obtained according to the above sample collection methods are also examined microscopically for cytological evidence of breast disease.

Description

15202530CA 02264277 1999-02-25WO 98/08976 PCTlUS97/ 14863Title: METHODS AND KITS FOR OBTAINING AND ASSAYING MAMMARYFLUID SAMPLES FOR BREAST DISEASES, INCLUDING CANCERTechnical Field: The invention relates to methods and kits for obtaining andassaying biological samples from mammary fluid. More specifically, the invention relatesto methods and kits for obtaining and assaying fluid and cytological samples from themammary glands of a mammalian subject for evaluating, diagnosing and managing breastdisease, including infections, pre-cancerous conditions, cancer susceptibility and cancer.BACKGROUND OF THE INVENTIONBreast cancer is by far the most common form of cancer in women, and is thesecond leading cause of cancer death in humans. Despite many recent advances indiagnosing and treating breast cancer, the prevalence of this disease has been steadilyrising at a rate of about 1% per year since 1940. Today, the likelihood that a womenliving in North America will develop breast cancer during her lifetime is one in eight.The current widespread use of mammography has resulted in improved detectionof breast cancer. Nonetheless, the death rate due to breast cancer has remained unchangedat about 27 deaths per 100,000 women. All too often, breast cancer is discovered at astage that is too far advanced, when therapeutic options and survival rates are severelylimited. Accordingly, more sensitive and reliable methods are needed to detect small (lessthan 2 cm diameter), early stage, in situ carcinomas of the breast. Such methods shouldsignificantly improve breast cancer survival, as suggested by the successful employmentof Papinicolou smears for early detection and treatment of cervical cancer.In addition to the problem of early detection, there remain serious problems indistinguishing between malignant and benign breast disease, in staging known breastcancers, and in differentiating between different types of breast cancers (eg. estrogendependent versus non-estrogen dependent tumors). Recent efforts to develop improvedmethods for breast cancer detection, staging and classification have focused on apromising array of so-called cancer “markers.” Cancer markers are typically proteins thatare uniquely expressed (eg. as a cell surface or secreted protein) by cancerous cells, or areexpressed at measurably increased or decreased levels by cancerous cells compared tonormal cells. Other cancer markers can include specific DNA or RNA sequences markingWO 98/089761015202530CA 02264277 1999-02-25PCT/US97/ 14863deleterious genetic changes or alterations in the patterns or levels of gene expressionassociated with particular forms of cancer.A large number and variety of breast cancer markers have been identified to date,and many of these have been shown to have important value for determining prognosticand/or treatment-related variables. Prognostic variables are those variables that serve topredict disease outcome, such as the likelihood or timing of relapse or survival.Treatment—related variables predict the likelihood of success or failure of a giventherapeutic plan. Certain breast cancer markers clearly serve both functions. Forexample, estrogen receptor levels are predictive of relapse and survival for breast cancerpatients, independent of treatment, and are also predictive of responsiveness to endocrinetherapy. Pertschuk et al., Cancer 66: 1663-1670, 1990; Parl and Posey, Hum. Pathol. _l2:960-966, 1988; Kinsel et al., Cancer Res. fig: 1052-1056, 1989; Anderson and PoulsonCancer Q: 1901-1908, 1989.The utility of specific breast cancer markers for screening and diagnosis, stagingand classification, monitoring and/or therapy purposes depends on the nature and activityof the marker in question. For general reviews of breast cancer markers, see Porter-Jordan et al., Hematol. Oncol. Clin. North Amer. §: 73-100, 1994; and Greiner,Pharmaceutical Tech., May, 1993, pp. 28-44. As reflected in these reviews, a primaryfocus for developing breast cancer markers has centered on the overlapping areas oftumorigenesis, tumor growth and cancer invasion. Tumorigenesis and tumor growth canbe assessed using a variety of cell proliferation markers (for example Ki67, cyclin D1 andproliferating cell nuclear antigen (PCNA)), some of which may be important oncogenes aswell. Tumor growth can also be evaluated using a variety of growth factor and hormonemarkers (for example estrogen, epidennal growth factor (EGF), erbB-2, transforminggrowth factor (TGF) or), which may be overexpressed, underexpressed or exhibit alteredactivity in cancer cells. By the same token, receptors of autocrine or exocrine growthfactors and hormones (for example insulin growth factor (IGF) receptors, and EGFreceptor) may also exhibit changes in expression or activity associated with tumor growth.Lastly, tumor growth is supported by angiogenesis involving the elaboration and growthof new blood vessels and the concomitant expression of angiogenic factors that can serveas markers for tumorigenesis and tumor growth.WO 98/08976l0I5202530CA 02264277 1999-02-25PCT/US97/14863In addition to tumorigenic, proliferation and growth markers, a number of markershave been identified that can serve as indicators of invasiveness and/or metastaticpotential in a population of cancer cells. These markers generally reflect alteredinteractions between cancer cells and their surrounding microenvironment. For example,when cancer cells invade or metastasize, detectable changes may occur in the expressionor activity of cell adhesion or motility factors, examples of which include the cancermarkers Cathepsin D, plasminogen activators, collagenases and other factors. In addition,decreased expression or overexpression of several putative tumor “suppressor” genes (forexample nm23, p53 and rb) has been directly associated with increased metastaticpotential or deregulation of growth predictive of poor disease outcome.In summary, the evaluation of proliferation markers, oncogenes, growth factorsand growth factor receptors, angiogenic factors, proteases, adhesion factors and tumorsuppressor genes, among other cancer markers, can provide important infonnationconcerning the risk, presence, status or future behavior of cancer in a patient.Determining the presence or level of expression or activity of one or more of these cancermarkers can aid in the differential diagnosis of patients with uncertain clinicalabnormalities, for example by distinguishing malignant from benign abnormalities.Furthermore, in patients presenting with established malignancy, cancer markers can beuseful to predict the risk of future relapse, or the likelihood of response in a particularpatient to a selected therapeutic course. Even more specific information can be obtainedby analyzing highly specific cancer markers, or combinations of markers, which maypredict responsiveness of a patient to specific drugs or treatment options.Methods for detecting and measuring cancer markers have been recentlyrevolutionized by the development of immunological assays, particularly by assays thatutilize monoclonal antibody technology. Previously, many cancer markers could only bedetected or measured using conventional biochemical assay methods, which generallyrequire large test samples and are therefore unsuitable in most clinical applications. Incontrast, modern immunoassay techniques can detect and measure cancer markers inrelatively much smaller samples, particularly when monoclonal antibodies thatspecifically recognize a targeted marker protein are used. Accordingly, it is now routineto assay for the presence or absence, level, or activity of selected cancer markers byimmunohistochemically staining breast tissue specimens obtained via conventional biopsy1015202530WO 98108976CA 02264277 1999-02-25PCT/US97/ 14863methods. Because of the highly sensitive nature of immunohistochemical staining, thesemethods have also been successfully employed to detect and measure cancer markers insmaller, needle biopsy specimens which require less invasive sample gatheringprocedures compared to conventional biopsy specimens. In addition, otherimmunological methods have been developed and are now well known in the art whichallow for detection and measurement of cancer markers in non-cellular samples such asserum and other biological fluids from patients. The use of these alternative samplesources substantially reduces the morbidity and costs of assays compared to proceduresemploying conventional biopsy samples, which allows for application of cancer markerassays in early screening and low risk monitoring programs where invasive biopsyprocedures are not indicated.For the purpose of breast cancer evaluation, the use of conventional or needlebiopsy samples for cancer marker assays is often undesirable, because a primary goal ofsuch assays is to detect the cancer before it progresses to a palpable or mammographicallydetectable tumor stage. Prior to this stage, biopsies are generally contraindicated, makingearly screening and low risk monitoring procedures employing such samples untenable.Therefore, there is general need in the art to obtain samples for breast cancer markerassays by less invasive means than biopsy, for example by semm withdrawal.Efforts to utilize serum samples for breast cancer marker assays have met withlimited success, largely because the targeted markers are either not detectable in serum, orbecause telltale changes in the levels or activity of the markers cannot be monitored inserum. In addition, the presence of breast cancer markers in serum probably occurs at thetime of micro—metastasis, making serum assays less useful for detecting pre-metastaticdisease. In contrast, fluid within the mammary glands themselves is expected to containmuch higher and more biologically relevant levels of breast cancer markers than serum,particularly in view of the fact that 80%-90% of all breast cancers occur within theintraductal epithelium of these glands. Fluid within the breast ducts is expected to containan assemblage and concentration of hormones, growth factors and other potential markerscomparable to those secreted by, or acting upon, the surrounding cells of the alveolar-ductal system. Likewise, mammary fluid is expected to contain cells and solid cellulardebris or products that can be used in cytological or immunological assays to evaluateWO 98/08976I015202530CA 02264277 1999-02-25PCT/US97/14863intracellular or cell surface markers that may not be detectable in the liquid fraction ofmammary fluid.Previous attempts to develop non-invasive breast cancer marker assays utilizingmammary fluid samples have included studies of mammary fluid obtained from patientspresenting with spontaneous nipple discharge. In one of these studies, conducted by Inajiet al., Qargzgr §_Q: 3008-3013, 1987, levels of the breast cancer marker carcinoembryonicantigen (CEA) were measured using conventional, enzyme linked immunoassay (ELISA)and sandwich-type, monoclonal immunoassay methods. These methods successfully andreproducibly demonstrated that CEA levels in spontaneously discharged mammary fluidprovide a sensitive indicator of nonpalpable breast cancer. In a subsequent study, also byInaji et al., Jpn. J. Clin. Oncol. _l2: 373-379, 1989, these results were expanded using amore sensitive, dry chemistry, dot-immunobinding assay for CEA determination. Thislatter study reported that elevated CEA levels occurred in 43% of patients tested withpalpable breast tumors, and in 73% of patients tested with nonpalpable breast tumors.CEA levels in the discharged mammary fluid were highly correlated with intratumoralCEA levels, indicating that the level of CEA expression by breast cancer cells is closelyreflected in the mammary fluid CEA content. Based on these results, the authorsconcluded that immunoassays for CEA in spontaneously discharged mammary fluid areuseful for screening nonpalpable breast cancer.Although the evaluation of mammary fluid has been shown to be a useful methodfor screening nonpalpable breast cancer in women who experience spontaneous nippledischarge, the rarity of this condition renders the methods of Inaji et al, inapplicable to themajority of women who are candidates for early breast cancer screening. In addition, thefirst Inaji report cited above determined that certain patients suffering spontaneous nippledischarge secrete less than 10 pl of mammary fluid, which is a critically low level for theELISA and sandwich immunoassays employed in that study. It is likely that otherantibodies used to assay other cancer markers may exhibit even lower sensitivity than theanti"-CEA antibodies used by Inaji and coworkers, and may therefore not be adaptable orsensitive enough to be employed even in dry chemical immunoassays of small samples ofspontaneously discharged mammary fluid.In view of the above, an important need exists in the art for more widely applicable,non-invasive methods and materials to obtain biological samples for use in evaluating,l0l5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863diagnosing and managing breast disease including cancer, particularly for screening earlystage, nonpalpable breast tumors. A related need exists for methods and materials thatutilize such readily obtained biological samples to evaluate, diagnose and manage breastdisease, particularly by detecting or measuring selected breast cancer markers, or panels ofbreast cancer markers, to provide highly specific, cancer prognostic and/or treatment-relatedinformation, and to diagnose and manage pre-cancerous conditions, cancer susceptibility,breast infections and other breast diseases.SUMMARY OF THE INVENTIONIt is therefore an object of the present invention to provide non-invasive methodsand kits for obtaining biological samples that can be employed in assays for evaluating,diagnosing and managing breast disease, particularly cancer.It is a further object of the invention to achieve the above object in assay methodsand kits that are widely applicable to a broad range of patients, and that include usefulassays and kits for screening early stage, nonpalpable mammary tumors.It is yet another object of the invention to provide methods and kits that utilize theaforementioned biological samples to evaluate, diagnose and manage breast disease,preferably breast cancer, by detecting and/or measuring selected breast disease markerssuch as breast cancer markers, or panels of breast cancer markers, to provide highlyspecific prognostic and/or treatment-related information to the clinician.The invention achieves these objects and other objects and advantages that willbecome apparent from the description which follows by providing non-invasive methodsfor obtaining biological samples from a mammary organ of a mammalian patient.Specifically, the methods of the invention involve administering oxytocin or an oxytocinanalog to a mammalian patient in an amount that is effective to stimulate expression ofmammary fluid from a nipple of the patient. The oxytocin is preferably administeredintranasally and is allowed to reach a target alveolar-ductal tissue of the breast where theoxytocin stimulates myoepithelial contraction of the alveolar-ductal tissue. Alternatively,an intramuscular or intravascular injection of oxytocin can effect the same myoepithelialcontraction response as the intranasal administration route. A mammary fluid collector,preferably a breast pump, is then applied to the nipple and is used to receive the expressedbreast fluid. In preferred methods involving use of a breast pump, negative pressure isWO 981089761015202530CA 02264277 1999-02-25PCT/US97/14863generated on the breast to facilitate the oxytocin stimulated expression of mammary fluid.Alternatively, the mammary fluid can be expressed and collected without the aid of abreast pump, which may require an increase of oxytocin dosage or lengthening of the postadministration time period before breast fluid is fully expressed from the nipple. Duringor after the mammary fluid expression step, a biological sample is collected from theexpressed mammary fluid, which sample may consist of whole mammary fluid, wholecells, cell fragments, cell membranes, selected liquid, cellular or other solid fractions ofthe mammary fluid, as well as proteins, glycoproteins, peptides, nucleotides (includingDNA and RNA polynucleotides) and other like biochemical and molecular constituents ofthe mammary fluid.In related aspects of the invention, methods are provided for determining thepresence or amount of a breast disease marker, preferably a breast cancer marker, inbiological samples obtained from a mammary organ of a mammalian patient. Thesemethods involve intranasal, intramuscular or intravascular administration of oxytocin oran oxytocin analog to mammalian patients in amounts effective to stimulate mammaryfluid expression in the patient. Once a sufficient post-administration time period haselapsed to allow the oxytocin to reach and stimulate target alveolar—ductal tissues,mammary fluid is collected directly from the nipple or, alternatively, the breast ispumped, and a biological sample from expressed mammary fluid is collected, as above.After the sample is collected a bioassay is conducted on the sample to determine thepresence and/or amount of the breast disease marker in the sample. Suitable bioassays inthis regard include assays to detect known markers of breast infection, such as assaysemploying immunological or other suitable probes to detect specific antigens and othermarkers expressed by selected pathogens, including bacterial and viral pathogens. Morepreferred bioassays will detect individual markers or panels of markers of benign breasttumors, pre-cancerous breast disease, and/or breast cancer, such as assays employingimmunological or other suitable probes to detect specific antigens and other markersexpressed by benign, pre-cancerous and/or cancerous alveolar—ductal cells of the breast.In yet additional aspects of the invention, clinically useful kits are provided fordetermining the presence and/or amount of a breast disease marker, preferably a breastcancer marker, in biological samples obtained from a mammary organ of a mammalianpatient. The kits include a pharmaceutical preparation of oxytocin in a biologically1015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863suitable carrier. Preferably, the oxytocin preparation is a solution of oxytocin provided inan intranasal spray applicator. The kits also preferably include a collecting device forcollecting a biological sample from the expressed mammary fluid, which collectingdevice may range from a simple fluid reservoir to solid phase media that can be directlyincorporated into solid phase bioassays. In this context, an optional breast pump may alsobe provided serving a dual purpose of applying negative pressure to the breast to facilitatemammary fluid expression from the nipple following oxytocin stimulation, and to providea reservoir or solid phase collecting device incorporated within the breast pump forbiological sample collection. In particularly preferred embodiments of the invention, kitsinclude compositions and/or devices for detecting the presence or amount of a breastdisease marker in the biological sample, for example an immunological or molecularprobe that binds or reacts with a breast cancer marker.DETAILED DESCRIPTION OF THE INVENTIONAs noted above, the invention provides methods for obtaining biological samplesfrom mammary fluid. Preferably, these methods are non-invasive, meaning they are non-surgical and do not involve penetration of the breast by needles or other intrusive devices.To achieve a non-invasive sample collecting method, the invention relies specifically onadministering the peptide hormone oxytocin to a mammalian patient, in an amount that iseffective to stimulate expression of mammary fluid from a nipple of the patient when abreast pump is applied to the nipple to assist the mammary fluid expression. Preferablythe oxytocin preparation is administered intranasally and is administered in an amountthat is intranasally effective to stimulate expression of mammary fluid from the nipple.Oxytocin is a peptide hormone of pituitary origin which is naturally released intothe bloodstream of lactating women in response to suckling, and stimulates contraction ofmyoepithelial cells in the mammary alveoli and ducts to cause milk ejection. Cobo. LPerinat. Med. g_l_: 77-85, 1993. The drug has also been widely used for stimulating laborin pregnant women, due to its activity of stimulating uterine contractions. Satin et al.,Am. J. Obstet. Gynecol. l66: 1260-1261, 1992. For these reasons, the pharmacology ofoxytocin has been thoroughly investigated, including detailed studies of effective dosages,half-life and potential side effects.WO 98/0897610I5202530CA 02264277 1999-02-25PCT/US97/14863For use in the present invention, an oxytocin preparation is provided for intranasal,intramuscular, or intravenous administration that contains oxytocin in a biologicallysuitable, liquid carrier. The most economic oxytocin preparations utilize a syntheticoxytocin analogue (eg. Pitocin® or Syntocinon®) available from various providers, forexample Sandoz (Basel, Switzerland) and United States Pharrnacopeia. Naturallyoccuring oxytocin from mammalian sources is of course suitable, as are other known,naturally occuring oxytocin-like peptide analogues and their synthetic counterparts havingsimilar activities for stimulating alveolar-ductal myoepithelial contraction. (See forexample, Amico et al., J. Clin. Endocrinol. Metab. §_Q: 5, 1985, incorporated herein byreference in its entirety).For use in the methods and kits of the invention, a preferred oxytocin preparationcontains approximately 40 USP units of oxytocin per ml of liquid carrier. Preferred liquidcarriers are biologically compatible solutions,isuch as a lactated Ringer's solution or otherphysiologically balanced, sterile, non-toxic and non—irritative solutions. To administer theoxytocin intranasally, a standard nasal squeeze bottle is used, which deliversapproximately 0.5 ml of the oxytocin preparation into the patient’s nostril when squeezed.The oxytocin is absorbed by the nasal mucosa into the systemic circulation where itreaches and acts specifically on the myoepithelial cells surrounding the alveoli of thebreast and making up the walls of the lactiferous ducts, causing their smooth musclefibers to contract and force any fluids present into the large ducts or sinuses where it canbe expressed from the nipple spontaneously onto a sample collector or by the furtheraction of a breast pump. Intranasal application of the spray preparation is therefore apractical and effective method of administration. The half-life of oxytocin in the humanbloodstream is extremely short, estimated to be about l0-l5 minutes or less, due to itsrapid removal from plasma by the kidney, liver, and mammary gland, and the time topharmacokinetic and clinical steady state is readily determined depending on the mode ofadministration (eg. bolus dosage, repeat administration, or steady infusion). (See forexample, Gonser, Arch. Gynecol. Obstet. 256: 63-66, I995; and Orhue, Obstet. Gynecol._8_§: 229-233, 1994, each incorporated herein by reference in its entirety). It is therefore aroutine matter to determine an appropriate concentration and dose of the oxytocinpreparation to administer an effective amount (either intranasally effective, intravenouslyeffective, or intramuscularly effective) of the oxytocin to cause expression of mammaryWO 9810897610202530CA 02264277 1999-02-25PCT/U S97/ 14863I 0fluid with or without the assistance of a breast pump. (See for example, Newton, i_<X_i_1_n_.N.Y. Acad. Sci. §5_2: 481-483; Mena, Neuroendocrinology Ql_: 722-730, 1995; Gonser,Arch. Gynecol. Obstet. 256: 63-66, 1995; Orhue, Obstet. Gynecol. _8_3_: 229-233, 1994;Satin et al., Am. J. Obstet. Gynecol., l66: 1260-1261, 1992; and Satin et al., QgsgGynecol. §§: 234-238, 1994, each incorporated herein by reference in its entirety).Although not all female patients are expected to be responsive to intranasaloxytocin stimulation, an intranasally effective amount of oxytocin for the purposes of theinvention can be readily determined. As used herein, an intranasally effective amount ofoxytocin is an amount of oxytocin sufficient to intranasally stimulate the expression of atleast 3 pl of mammary fluid in at least 50% of non-lactating female patients with the aidof negative pressure to the nipple of between 50-200 mm Hg applied by a breast pump upto 45 min after a first administration of the oxytocin spray. It may be necessary, andindeed preferred, to administer a low, preliminary dose of oxytocin to the patient, forexample a single spray of a 40 Unit/ml oxytocin solution in each nostril, or multiplesprays of a lower concentration oxytocin preparation, and thereafter wait to determine aparticular patient’s sensitivity. If there is no reaction with an initial application of thebreast pump after a short post-administration period of 2-15 minutes, and preferably 2-5minutes, a booster dose of the oxytocin spray may be administered and the pumpreapplied. In this way, the clinician can modulate the dosage to each patient’s varyingsensitivity, and thereby minimize potential adverse side effects. Alternatively,intramuscular or intravenous ocytocin administration can be used according to the samedosage determination and administration principles in patients where intranasaladministration fails or is otherwise contra-indicated as a preferred mode of administration.Once an intranasally effective dose of the oxytocin is administered and theclinician has allowed a suitable post-administration period to elapse for the oxytocin toreach and stimulate the target alveolar-ductal tissue, the breast pump is applied accordingto well known procedures and negative pressure is generated on the breast to facilitate theexpression of mammary fluid. Within the methods of the invention, negative pressures of50-200 mm Hg are preferred, and these pressures are maintained, preferablyintermittently, for approximately 1-15 minutes, depending on the sensitivity of individualpatients, oxytocin dosage and other factors. Alternatively, mammary fluid expression canWO 98/089761015202530CA 02264277 1999-02-25I 1 PCT/US97/14863be achieved without the aid of a breast pump using a simple collector to receive theexpressed breast fluid, as described herein.The volume of expressed mammary fluid will vary depending on a variety offactors, including patient sensitivity to oxytocin, dosage of oxytocin delivered, time andpressure of breast pump administration, and other factors. For the least sensitive breastmarker assays of the invention, a volume of expressed mammary fluid of 300-500 ul ispreferred to provide ample material for conducting the assay, and these volumes will beobtainable from a substantial proportion of women treated according to the abovemethods. To express 300-500 ul of mammary fluid, some women will require repeatedstimulation treatments, perhaps requiring pooling of mammary fluid samples obtainedduring multiple patient visits. However, for more sensitive assays of the invention, eg.solid phase immunoassays, much smaller samples of 3 pl or less will be suitable to carryout the assays, particularly in the case of breast cancer markers that are naturally secretedinto the mammary fluid and are therefore expected to be present in very highconcentrations compared to, for example, breast epithelial cell surface antigens orintracellular antigens that are not secreted.During or after the mammary fluid expression step, a biological sample iscollected from the expressed mammary fluid. A range of suitable biological samples arecontemplated and will be useful within the methods of the invention, including wholemammary fluid, selected liquid or solid fractions of the mammary fluid, whole cells orcellular constituents, proteins, glycoproteins, peptides, nucleotides (including DNA andRNA polynucleotides) and other like biochemical and molecular constituents of themammary fluid. Sample collection can be achieved simply by receiving the expressedmammary fluid within a suitable reservoir, such as an ordinary sample storage containeror assay vessel. Alternatively, samples can be collected by exposing the expressedmammary fluid to conventional buffers, diluents, extraction or chromatographic media,filters, etc., to stabilize or prepare the sample for further processing or direct incorporationinto a desired assay. In preferred embodiments of the invention, the expressed mammaryfluid is exposed to a solid phase medium, such as a microscopic glass slide, nitrocellulosefilter, affinity column, dot blot matrix or other like medium, that will selectively adsorb,bind, filter or otherwise process desired components of the mammary fluid, such as bulkor selected proteins, for convenient incorporation into a desired assay. The range of l015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486312contemplated sample collection procedures and materials that are useful within theinvention is broad, and selected methods and materials will vary with each selected assay,as will be understood and readily practiced by those skilled in the art.Although a fundamental utility of the present invention lies in the novel, non-invasive methods for obtaining biological samples from mammary fluid, additionalmethods are disclosed herein that provide useful assays for detecting and/or measuringimportant breast disease markers in these samples. In this context, the invention providesa broad range of assay methods incorporating known procedures and reagents fordetennining the presence and/or expression levels of breast disease markers, particularlybreast cancer markers, in biological samples. As incorporated within the invention, thesemethods involve administration of oxytocin to mammalian patients, preferably viaintranasal administration, in amounts effective to stimulate mammary fluid expression inthe patient, as described above. Once a sufficient post-administration time period haselapsed to allow the oxytocin to reach and stimulate target alveolar—ductal tissues, thebreast is pumped and a biological sample is collected, as described above. After thesample is collected, a bioassay is conducted on the sample to determine the presenceand/or amount of a selected breast disease marker, preferably a breast cancer marker orpanel of breast cancer markers, in the sample.As used herein, the term breast disease marker refers to any cell, cell fragment,protein, peptide, glycoprotein, lipid, glycolipid, proteolipid, or other molecular orbiological material that is uniquely expressed (eg. as a cell surface or secreted protein) bydiseased breast cells, or is expressed at a statistically significant, measurably increased ordecreased level by diseased breast cells, or in association with breast disease (eg. a proteinexpressed by an infectious agent associated with breast disease), or is expressed at astatistically significant, measurably increased or decreased level by diseased breast cellscompared to normal breast cells, or which is expressed by non-diseased breast cells inassociation with breast disease (eg. in response to the presence of diseased breast cells orsubstances produced therefrom). Breast disease markers can also include specific DNAor RNA sequences marking a deleterious genetic change, or an alteration in patterns orlevels of gene expression significantly associated with breast disease. Preferred breastdisease markers include markers of breast infections, benign neoplasia, malignantCA 02264277 1999-02-25WO 98108976 13 PCT/US97/ 14863neoplasia, pre-cancerous conditions, and conditions associated with an increased risk ofcancenAs used herein, the term breast cancer marker refers to a subset of breast diseasemarkers, namely any protein, peptide, glycoprotein, lipid, glycolipid, proteolipid, or other5 molecular or biological material that is uniquely expressed (eg. as a cell surface orsecreted protein) by cancerous cells, or is expressed at a statistically significant,measurably increased or decreased level by cancerous cells compared to normal cells, orwhich is expressed by non—cancerous cells in association with cancer (eg. in response tothe presence of cancerous cells or substances produced therefrom). Breast cancer markers10 can also include specific DNA or RNA sequences marking a deleterious genetic change,or an alteration in patterns or levels of gene expression significantly associated withcancer. In addition, breast cancer markers can include cytological features of whole cellspresent in mammary fluid, such as nuclear inclusions or cytoplasmic structures or stainingattributes uniquely expressed by, or associated with, cancerous cells.15 Among the breast cancer markers that are useful within the methods of theinvention, a subset are described in representative review articles by Porter-Jordan et al.,Hematol. Oncol. Clin. North Amer. §: 73-100, 1994; and Greiner, Pharmaceutical Tech,May, 1993, pp. 28-44, each incorporated herein by reference in its entirety. Other suitablemarkers are also widely known and can be readily incorporated into the methods of the20 invention using information and methods generally known or available in the literature.Preferred breast cancer markers for use within the invention include well characterizedmarkers that have been shown to have important value for determining prognostic and/ortreatment—related variables in human female patients. As noted previously, prognosticvariables are those variables that serve to predict outcome of disease, such as the25 likelihood or timing of relapse or survival. Treatment-related variables predict thelikelihood of success or failure of a given therapeutic program. Determining the presenceor level of expression or activity of one or more of these markers can aid in thedifferential diagnosis of patients with malignant and benign abnormalities, and can beuseful for predicting the risk of future relapse or the likelihood of response to a selected30 therapeutic option.It is important to note, however, that the invention does not rely solely on breastdisease markers that meet the stringent requirements of sensitivity and specificity that l0202530WO 98/08976CA 02264277 1999-02-2514 PCT/U S97/ 14863would render the marker immediately acceptable for clinical application to humanpatients. On the contrary, a number of breast disease markers contemplated within theinvention fall short of these stringent criteria, and nonetheless provide useful informationthat can be of substantial benefit in detecting, differentially diagnosing or managing breastcancer. Such non—clinically accepted markers are useful for immediate application withinthe methods of the invention as basic research tools, and as adjunctive tools in clinicalapplications. Beyond these immediate applications, many such markers are expected tobe further developed and refined according to the methods of the invention to the point ofdirect clinical applicability, particularly in assay methods that analyze combinations ofmarkers to generate complementary data of greater predictive value than data yielded byindividual markers alone.The preferred assay methods of the invention particularly focus on breast cancermarkers associated with tumorigenesis, tumor growth, neovascularization and cancerinvasion, and which by virtue of this association provide important informationconcerning the risk, presence, status or future behavior of cancer in a patient. As notedpreviously, tumorigenesis and tumor growth can be assessed using a variety of cellproliferation markers (for example Ki67, cyclin D1 and PCNA). Tumor growth can alsobe evaluated using a variety of growth factor and hormone markers (for example estrogen,EGF, erbB-2, and TGF (X), receptors of autocrine or exocrine growth factors andhormones (for example IGF and EGF receptors), or an giogenic factors. In addition totumorigenic, proliferation and growth markers, a number of markers provide informationconcerning cancer invasion or metastatic potential in cancer cells, for example byindicating changes in the expression or activity of cell adhesion or motility factors.Exemplary markers in this context include Cathepsin D, plasminogen activators andcollagenases. In addition, expression levels of several putative tumor “suppressor" genes,including nm23, p53 and rb, provide important data concerning metastatic potential, orgrowth regulation of cancer cells. A large number and variety of suitable breast cancermarkers in each of these classes have been identified, and many of these have been shownto have important value for determining prognostic and/or treatment-related variablesrelating to breast cancer.Prior to or concurrent with each assay run of the invention, it is preferable toperform a preliminary evaluation to verify sample origin and/or quality. The focus ofl0l5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863l 5such preliminary evaluations is to verify that the sample collected from expressedmammary fluid is indeed of mammary origin, and is not contaminated with other potentialcontaminants, such as sweat from skin surrounding the nipple. For these sampleverification purposes, a variety of assays are available which identify mammary fluidmarkers known to be present in mammalian mammary fluid, and which are preferablyhighly specific markers for mammary fluid (i.e. markers which are typically alwayspresent in mammary fluid and which are absent from all, or most of, other potentiallycontaminating bodily fluids and tissues). However, an acceptable level of specificity formammary fluid markers within the methods of the invention is provided by markers thatare simply known to be present in mammary fluid, even though they may be present inother bodily fluids. One such marker is the enzyme lysozyme, which is a normalcomponent of human serum, urine, saliva, tears, nasal secretions, vaginal secretions,seminal fluid, and mammary fluid. Lysozyme (muramidase) is an enzyme whichhydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharide cell wall of a varietyof microorganisms resulting in cell lysis. Quantitative measurement of lysozyme isreadily accomplished by a well known agar plate diffusion method, described in detail inthe instructions provided with the Quantiplate® lysozyme test kit, available fromKallestad, Sanofi Diagnostics (Chasta, MN), incorporated herein by reference in itsentirety. 9Other mammary fluid markers for sample verification that are more specific thanlysozyme are preferred within the methods of the invention, and can be readilyincorporated within the invention based on published and generally known information.The most preferred among these markers are proteins and other biological substances thatare specifically expressed or enriched in mammary fluid. A diverse array of suitablemarkers in this context have been characterized and have already been used to developspecific antibodies, including affinity purified and monoclonal antibodies. Theseantibodies can in turn be employed as immunological probes to determine the presence orabsence, and/or to quantify, selected mammary fluid markers to verify mammary fluidsample origin and quality. Mammary fluid markers of particular interest for use withinthe invention include specific cytokeratins that are characteristically expressed by normaland cancerous mammary epithelial cells, against which specific panels of antibody probeshave already been developed. (See for example, Nagle, J. Histochem. Cytochem. $2 869-10I5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863l 6881, 1986, incorporated herein by reference in its entirety). Also useful as mammary fluidmarkers are the human mammary epithelial antigens (HME-Ags) corresponding toglycoprotein components of the human milk fat globulin (HMFG) protein, against whichspecific antibodies (eg anti HMFG1, Unipath, U.K.) are also available. (see Rosner et al.,Cancer Invest. Q: 573-582, 1995; Ceriani et al. Proc. Natl. Acad. Sci. USA 23: 582-586,1982; Ceriani et al., Breast Cancer Res. Treat. _1_§; 161-174, 1990, each incorporatedherein by reference in its entirety).To conduct the breast disease marker assays provided within the invention, acollected biological sample from mammary fluid is generally exposed to a probe thatspecifically binds to a selected breast disease or breast cancer marker, or otherwiseinteracts with the marker in a detectable manner to indicate the presence or absence, oramount, of the breast disease or breast cancer marker in the sample. Selected probes forthis purpose will generally depend on the characteristics of the breast disease marker, i.e.on whether the marker is a protein polynucleotide or other substance. In preferredembodiments of the invention, the breast disease marker is a protein, peptide orglycoprotein, all of which are effectively targeted in breast disease marker assays usingspecific immunological probes. These immunological probes can be labeled with acovalently bound label to provide a signal for detecting the probe, or can be indirectlylabeled, for example by a labeled secondary antibody that binds the immunological probeto provide a detectable signal.General methods for the production of non—human antisera or monoclonalantibodies (e.g., murine, lagonnorpha, porcine, equine) are well known and may beaccomplished by, for example, immunizing an animal with a selected breast diseasemarker protein, peptides synthesized to include part of the marker protein sequence,degradation products including part of the marker protein sequence, or fusion proteinsincluding all or part of the marker protein linked to a heterologous protein or peptide.Within various embodiments, monoclonal antibody producing cells are obtained fromimmunized animals, immortalized and screened, or screened first for the production of anantibody that binds to the selected breast cancer marker protein or peptide, and thenimmortalized. It may be desirable to transfer the antigen binding regions (i.e., F(ab')2 orhypervariable regions) of non—human antibodies into the framework of a human antibodyby recombinant DNA techniques to produce a substantially human molecule. Methodsl0I5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863l 7for producing such "humanized" molecules are generally well known and described in, forexample, U.S. Patent No. 4,816,397 (incorporated herein by reference in its entirety).Alternatively, a human monoclonal antibody or portions thereof may be identified by firstscreening a human B—cell CDNA library for DNA molecules that encode antibodies thatspecifically bind to the selected breast disease marker according to the method generallyset forth by Huse et al. ( gig: 1275-1281, 1989 (incorporated herein by referencein its entirety). The DNA molecule may then be cloned and amplified to obtain sequencesthat encode the antibody (or binding domain) of the desired specificity.Also contemplated within the invention are bifunctional antibodies havingindependent antigen binding sites on each immunoglobulin molecule (as disclosed forexample in Thromb. Res. Suppl. 2(_: 83, 1990, and in The Second Annual IBCInternational Conference on Antibody Engineering, A. George ed., Dec. I6-18, 1991;each incorporated herein by reference in its entirety), as well as panels of individualantibodies having differing specificities. Bifunctional antibodies and antibody panels ofparticular use within the invention include antibodies and panels of antibodies that bind totwo or more selected breast disease markers to generate complementary data of greaterpredictive value than data yielded by individual markers alone.Monoclonal antibodies are particularly useful within the invention as labeledprobes to detect, image and/or quantify the presence or activity of selected breast diseasemarkers. In this context, monoclonal antibodies that specifically bind to selected breastdisease markers are provided which incorporate one or more well known labels, such as adye, fluorescent tag or radiolabel. By incorporating such a label, the antibodies can beemployed in routine assays to detennine expression, localization and/or activity of one ormore selected breast disease markers in a biological sample including, or derived from,mammary fluid. Results of these assays to determine expression, localization and/oractivity of a selected breast disease marker in a test sample taken from a patient at risk forbreast disease, or known to have breast disease, can be compared to results from controlstudies detecting and/or quantifying the same marker in biological samples obtained fromnormal patients negative for breast disease. In this manner, baseline data and cutoffvalues can be determined according to routine methods to refine the assays of theinvention and adapt them for direct clinical application.15202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863I 8Detection and/or quantification of breast disease markers in the biological samplesof the invention can be accomplished using a variety of methods. Preferred methods in thisregard include well known ELISA immunoassays, immunoprecipitation assays, and varioussolid phase immunoassays including Western blotting, dot blotting and affinity purificationimmunoassays, among other methods. Comparable methods are disclosed herein, or areelsewhere disclosed and known in the art, for using non-antibody probes to detect and/orquantify the expression and/or activity of breast disease markers. Suitable non-antibodyprobes for use within the invention include, for example, labeled nucleotide probes thathybridize at standard or high stringency to DNA transcripts of oncogenes and other DNAsequences associated with elevated breast disease risk, or with mRNA transcripts encodingbreast disease marker proteins. Other suitable probes include labeled ligands, bindingpartners and co-factors of breast disease markers (eg. growth factor receptor ligands, orsubstrates of breast cancer associated proteases such as cathepsin D). In certain preferredembodiments of the invention, cDNA and oligonucleotide probes are employed inNorthern, Southern and dot—blot assays for identifying and quantifying the level ofexpression of a selected breast disease marker in cell samples collected from expressedmammary fluid. Measuring the level of expression of breast disease markers according tothese methods will provide important prognostic and treatment-related information forassessing a broad range of breast disease, including the genesis, growth and invasiveness ofcancer, in mammals, particularly humans. For example, assays utilizing oligonucleotideprobes will assist early screening to evaluate heritable genetic lesions associated with breastcancer, and to distinguish between pre-cancerous, early cancerous and likely metastaticlesions in patients.In addition to the above mentioned sample collection and assay methods, theinvention also provides kits and multicontainer units comprising reagents and componentsfor practicing the sample collection and assay methods of the invention. Briefly, thesekits include basic components for obtaining a biological sample from mammary fluid,including a pharmaceutical preparation of oxytocin in a biologically suitable carrier.Preferably, the oxytocin preparation is provided in an intranasal spray applicator andcontains approximately 40 USP units of oxytocin per ml of liquid carrier, which carrier isa simple, inexpensive buffered saline solution. Preferred applicators can be in any of avariety of pressurized aerosol or hand—pump reservoir forms, with a nozzle for directing a 1015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863l 9liquid spray of the oxytocin into a patient’s nostril. The kits also preferably include acollecting device for collecting a biological sample from the expressed mammary fluid,which collecting device may range from a simple fluid reservoir to solid phase media thatcan be directly incorporated into solid phase bioassays. In this context, an optional breastpump may also be provided that is applicable to a human breast and designed to generateintermittent or sustained negative pressures in an area surrounding the nipple of betweenabout 50-200 mm Hg. More preferably, the breast pump serves a dual purpose ofapplying negative pressure to the breast to facilitate mammary fluid expression from thenipple following oxytocin stimulation, and to provide a reservoir or solid phase collectingdevice incorporated within the breast pump for biological sample collection.Kits for practicing the assay methods of the invention include a suitable containeror other device for collecting a biological sample from expressed mammary fluid. Arange of suitable collection devices are contemplated corresponding to a wide range ofsuitable biological samples that may be collected from the expressed mammary fluid. Forexample, simple sterile containers or reservoirs are provided to collect whole mammaryfluid. Alternatively, a variety of solid phase devices, including microscopic glass slides,membranes, filters, beads and like media, are provided to receive or partition selectedliquid or solid fractions of the mammary fluid, to receive or partition cells or cellularconstituents from the mammary fluid, or to receive or partition purified or bulk proteins,glycoproteins, peptides, nucleotides (including DNA and RNA polynucleotides) or otherlike biochemical and molecular constituents from the mammary fluid. A wide variety ofsuch sample collection devices are disclosed herein, or are otherwise widely known ordescribed in the literature, which can be readily adapted for use within specificembodiments of the invention. These collection devices may be provided as a componentof the breast pump (such as a removable fluid reservoir or nitrocellulose filter placedwithin the pump to directly receive or contact the expressed mammary fluid as it ispumped), or may be provided separately (for example as a non—integral membrane, filter,affinity column or blotting material to which mammary fluid or mammary fluidcomponents are exposed to collect a biological sample for assay purposes).In more detailed embodiments of the invention, kits include reagents and/ordevices for detecting the presence and/or amount of a breast disease marker in thebiological sample, for example an immunological or molecular probe that binds or reactsI015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486320with a breast cancer marker. Among these possible reagents or devices areimmunological and non-immunological probes for detecting the presence or amount of abreast cancer marker in the biological sample. The kits may also contain suitable buffers,preservatives such as protease inhibitors, direct or sandwich-type labels for labeling theprobes, and/or developing reagents for detecting a signal from the label. In one aspect,kits of the present invention contain monoclonal antibodies useful for detecting and/ormeasuring a breast cancer marker in a sample. Such antibodies may be pre-labeled, ormay be detected by binding to a secondary antibody optionally included in the kit. Theantibody reagents may be provided in a separate container, or may be provided incombination in a series of containers. Within yet another aspect of the invention, kitscontain sequence-specific oligonucleotide primers for detecting polynucleotide moleculesencoding breast cancer marker proteins. Such primers may be provided in separatecontainers, or may be provided in combinations of one or more primer pairs in a series ofcontainers. A broad selection of other kits are provided within the invention based ongeneral knowledge in the art and on the description herein, including kits that containspecific instructions for carrying out the assays of the invention.The following examples are offered by way of illustration, not by way oflimitation.EXAMPLE 1Stimulation of Mammary Fluid Expression by lntranasal Administration of OxytocinCoupled With Breast Pump ApplicationOxytocin nasal solution, acts specifically on the myoepithelial elementssurrounding the alveoli of the breast and making up the walls of the lactiferous ducts,causing their smooth muscle fibers to contract and thus force any fluids present into thelarge ducts or sinuses where it can be expressed from the nipple by the further action of abreast pump. The nasal spray is promptly absorbed by the nasal mucosa to enter thesystemic circulation. Intranasal application of the spray preparation is a practical andeffective method of administration. Half-life of oxytocin in the human circulation isextremely short, approximately 10-15 minutes, and oxytocin is then rapidly removed fromplasma by the kidney, liver, and mammary gland.l0l5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/14863g 21Because of the known effects of oxytocin to cause uterine contractions, pregnantwomen should not be treated by the methods contained herein unless the benefits oftesting outweigh the risk of inducing premature labor.The oxytocin nasal solution contains a concentration of natural or syntheticoxytocin, or a functional analog thereof, that is intranasally effective in a selected volumeof administered spray to stimulate expression of mammary fluid from a nipple of thepatient when a breast pump is applied to the nipple to assist mammary fluid expression.In the present example, a preferred oxytocin preparation containing approximately 40USP units of oxytocin per ml of lactated Ringer’s solution is administered into the nosewith the squeeze bottle held in the upright position while the patient is in a sittingposition. One or two sprays are administered into each nostril from a standard nasalsqueeze bottle, which delivers approximately 0.5 ml of the oxytocin solution per spray ina fine mist when the bottle is squeezed. The number and volume of sprays administered,as well as the concentration of oxytocin in the solution, can be adjusted according to wellknown pharrnacokinetic principles (See for example, Newton, Ann. N.Y. Acad. Sci. 652:481-483; Mena, Neuroendocrinology _6__l: 722-730, 1995; Gonser, Arch. Gynecol. Obstet.Q6: 63-66, 1995; Orhue, Obstet. Gynecol. §3: 229-233, 1994; Satin et al., Am. J. Obstet.Gynecol., _1_6_§: 1260-1261, 1992; and Satin et al., Obstet. Gynecol. §§: 234-238, 1994,each incorporated herein by reference in its entirety) to ensure that the amount of oxytocinadministered to the patient corresponds to an intranasally effective amount to stimulatethe expression of at least 3 pl of mammary fluid in at least 50% of non-lactating femalepatients with the aid of the breast pump. For example, adjustments may be desired in thenumber of sprays delivered to the patient, and/or the timing of spray delivery, so that theclinician can modulate the dosage to each patient’s varying sensitivity, and therebyminimize potential adverse side effects. In the present example, a preliminary dose of asingle spray of the 40 Unit/ml oxytocin solution is delivered into each nostril of thepatient, and the administering clinician waits for a short post-administration period of 2-3minutes. After this period the breast pump is applied, and the clinician determineswhether or not the amount of oxytocin delivered was sufficient to stimulate breast pumpassisted expression of mammary fluid. If no fluid is expressed at this stage a booster doseof 1 or 2 additional sprays of the oxytocin solution is administered in each nostril, and thepump is reapplied after a 5-10 minute post-booster administration period.l0l5202530CA 02264277 1999-02-25wo 93/03975 PCT/US97/1486322After the intranasally effective dose of the oxytocin is administered and theclinician has allowed a suitable post-adminsitration period to elapse for the oxytocin toreach and stimulate the target alveolar-ductal tissue, the breast pump is applied accordingto well known procedures. Negative pressures of 50-200 mm Hg are applied in the areaof the nipple and are maintained, intermittently or continuously, for approximately 1-15minutes, depending on the sensitivity of individual patients, oxytocin dosage and otherfactors. Alternatively, oxytocin can be administered by intramuscular or intravascularroutes by well known means (Oxytocin Injection (synthetic), USP; Wyeth—AyerstLaboratories) to effect the same response as intranasal administration.Using the above methods, primary samples of mammary fluid containing at least 3til of fluid are expressed by 50% or more of non-lactating female patients. During or afterthe mammary fluid expression step, a biological sample is collected from the expressedmammary fluid. In the present example, a nitrocellulose filter is placed within the breastpump in line with a path of the expressed mammary fluid into the pump, so that theexpressed fluid contacts the filter. Upon contact of the primary sample of expressedmammary fluid with the filter, cells, proteins and other desired components of the\ mammary fluid adhere to the filter to form a filter—bound biological sample for subsequentanalysis. Other suitable biological samples, including whole mammary fluid samples,cytological samples of whole cells, membranes or other cellular components, and samplescontaining proteins, glycoproteins, peptides, nucleotides and other constituents of theprimary mammary fluid sample can be collected with appropriate modifications of theabove procedures, according to well known principles and methods.EXAMPLE 2Verification of Sample Origin and Quality Using Lvsozvme AnalysisTo ascertain that the primary sample of mammary fluid, or the collected sample,obtained by the above methods is of mammary origin and is not corrupted by likelycontaminants, one or more constituents of normal mammary fluid are assayed for. In thepresent example, an enzyme that is ordinarily present in mammary fluid, lysozyme, isassayed in the primary mammary fluid sample to help confirm that the sample is ofmammary origin. Lysozyme (muramidase) is an enzyme which hydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharide cell wall of a variety of microorganisms,1015202530WO 98108976CA 02264277 1999-02-25PCT/US97/1486323which activity can be readily detected and quantified using a routine, inexpensive assay.In the present example, Lysozyme is measured in the primary mammary fluid sampleusing the Quantiplate Lysozyme Test kit (Kallestad, Chasta, MN). The assay employs thereagents and procedures provided by the manufacturer and specified in detail in themanufacturer’s instructions, with the exception that a mammary fluid sample issubstituted in place of serum, urine or tears. Analysis of these results establishes that thesample contains lysozyme, which is a normal component of human serum, urine, saliva,tears, nasal secretions, vaginal secretions, seminal fluid, and mammary fluid.More specific assays are used in place of the lysozyme assay, or to supplementlysozyme assay results, particularly where clinical data for human patients are beinggathered. Other mammary fluid markers for sample verification that are more specificthan lysozyme can be readily incorporated within the invention, based on published andgenerally known information. In one example, the presence of cathepsin D is assayedusing the monoclonal antibodies and methods disclosed in Vetvicka et al., Biochem. Mol.Biol. Int’l. _3__Q: 921-928, 1993, incorporated herein by reference in its entirety). In anotherexample, one or more human mammary epithelial antigens (HME—Ags) corresponding toglycoprotein components of the human milk fat globulin (HMFG) protein are detected inthe primary mammary fluid sample, or in the biological sample that is used in the breastcancer marker assay, using specific antibody probes, as described by Rosner et al., §_a_r£;rI_nvLst.1§: 573-582, 1995; Ceriani et al. Proc. Natl. Acad. Sci. USA 141: 582-586, 1982;Ceriani et al., Breast Cancer Res. Treat. l_5_; 161-174, 1990, each incorporated herein byreference in its entirety). In many cases, the sample verification assay can be incorporatedwithin the breast cancer marker assay in a single procedure, for example as describedbelow in Example 4, an assay for HME-Ags (wherein the HME-Ag findings are indicativeof sample origin/quality, and also of the presence and/or quantity of a specific breastcancer marker in the sample). In another example, sample verification is achievedthrough a combinatorial (i.e. multiple marker) immunoassay targeting variouscytokeratins, which can be detected as a panel of cytokeratins specifically expressed inmammary tissue sample. (See, Nagle, J. Histochem. Cytochem. _3_4_: 869-881, 1986,incorporated herein by reference in its entirety). One or more of these cytokeratins (eg.K5, K8, K18 and K19) can be simultaneously or independently measured in the context ofa breast cancer assay, and the level of expression of the subject cytokeratin(s) can yield.. .......s........................... .. ., .. . .. ..A....”.....m................... I5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/ 1486324information concerning the presence or status of breast cancer in a patient. (See forexample, Focus, Harvard University News Office, March 21, 1991, pp. 2-3; and Lee,Proc. Natl. Acad. Sci. 8_8: 1991, each incorporated herein by reference in its entirety).EXAMPLE 3Cytology in Biological Samples From Mammary FluidThis example describes the use of conventional cytological techniques to identifyand classify breast diseases from samples obtained as described in Example 1. Followingcollection of the sample in the sample collector, the central region of a clean glassmicroscopic slide is touched to the sample and a cover slip is slid over the sample tospread it along the surface of the slide. The slide is allowed to air dry and then is fixed, forexamample in absolute alcoho,l and stained with standard cytological stains, such asmethylene blue, hematoxylm and rosin, and other suitable stains.The slides are then examined by light microscopy for evidence of atypical growthof cells and clumps of cells, using well known methods, including those described in"Diagnosis of Non-Palpable Breast Lesions: Ultrasonographically Controlled Fine-NeedleAspiration: Diagnostic and prognostic Implications of Cytology" by JacquelineMouriquand, published by S Karger Pub: July 1993, ISBN: 3805557477; "Breast: Guidesto Clinical Aspiration Biopsy" by Tilde S. Kline, Irwin K. Kline, published by Igaku—Shoin Medical Pub: May 198g, LSBN: 0896401596; "Cytopathology ofthe Breast (AsopTheory and Practice of Cytopathology; 5)" by Shahla Masood, published by AmericanSociety of Clinical Pathology: November l99S, ISBN: 0891893806; "Fine NeedleAspiration Cytology and Its Clinical Applications: Breast and Lung" by Philip S.Feldman, published by American Society of Clinical Pathology: November 1984, ISBN:0891891846, each incorporated herein by reference in its entirety.EXAMPLE 4Immunoassay for Human Mammary Epithelial Antigens in Biological Samples FromMammary FluidHuman mammary epithelial antigens (HME-Ags) are glycoprotein components ofthe human milkfat globule (HMFG) and of the membrane of the breast epithelial cell,which are released by breast tumors and not by normal breast tissue. (Ceriani et al., Proc.. A» .. . .,.....m..W1,..,1,......e._.....-,. , ,10I5202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486325Natl. Acad. Sci. _7_4: 582-586, 1977, incorporated herein by reference in its entirety). Inthe present example, several HME-Ags, having molecular weights of I50, 70, and 45kilodaltons, are detected and measured using specific anti-HMFG or anti—humanmammary epithelial (ot-HME) probes prepared and employed as described by Ceriani etal., Proc. Natl. Acad. Sci. _7_9: 5420~5425, 1982 (incorporated herein by reference in itsentirety).To begin the assay, biological samples from mammary fluid collected onnitrocellulose filters are eluted electrophoretically into phosphate buffered saline toprovide a test sample, according to standard methods. Alternatively, whole mammaryfluid or other types of biological samples obtained from mammary fluid can beconstituted in an appropriate medium or mixture to provide a test sample for the assay.Once the test sample is thus provided, it is then assayed according to the HME-Agsradioimmunoassay (RIA) methods described in Ceriani et al., Breast Cancer Res. Treat.l_5_: 161-174, 1990 (incorporated herein by reference in its entirety).Briefly, the RIA includes two preliminary treatments of the biological samples toseparate interfering factors: a centrifugation step to separate out any fat present, and asecond, precipitation step to precipitate potential immunocomplexes usingpolyethyleneglycol (PEG). The next steps comprise the assay proper, where HMFGantigen bound to a solid support (microtiter plates) is presented to stoichiometric or lesseramount of the oi-HME antibody probe, and binding of the ot—HME is competed by thebiological samples from mammary fluid preliminarily treated as above. The amount of ot-HME bound to HMFG antigen on the solid phase is determined in a final step bydetection of the ot-HME antibody probe by radioiodinated, affinity-purified rabbit anti-mouse immunoglobulin.Solutions used in the assay are as follows: i) Phosphate buffered saline (PBS):176 ml 0.05M KH2PO4, 608 ml 0.05M NagHPO4, and 8 g NaCl brought up to 1000 ml inH20 (pH7.4). ii) RIA buffer: 0.1% BSA, in 0.3% Triton—X-100 (Research Prod.International Corp., Mount Prospect, IL) plus 0.05% sodium azide in PBS. iii) Detergentbuffer: 0.3% Triton-X-100 plus 0.05% sodium azide in PBS. iv) Buffered polyethyleneglycol (PEG): 6.6% PEG (M.W. 8000) (Sigma) plus 0.05% sodium azide in PBS) '51-labeled affinity—purified rabbit anti-mouse immunoglobulin (Rot-mouse lg) (Antibodies,Inc., Davis, CA), radioiodinated by the chloramine—T procedure as reported (Ceriani et al., 1015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486326Proc. Natl. Acad. Sci. 19: 5420-5425, 1982) and diluted to 4x106 cprn/ml, in RIA buffer.Rabbit polyclonal anti-HMFG antibodies or rabbit anti-human mammary epithelialantibodies (0t—HME) were prepared and assayed as described (Id.).To prepare a standard curve for evaluating assay results, control samples fromnormal human mammary fluid (exposed to nitrocellulose filters and eluted in the samemanner as the nitrocellulose adsorbed, eluted test sample, or alternatively provided asnormal whole mammary fluid or other selected type of sample obtained from normalmammary fluid, constituted in an appropriate medium or mixture to provide a suitablecontrol assay sample) are centrifuged for 7 min at 10,240 rpm at 10 ° C. The upper whiteband formed at the top of the sample (if there is one) is discarded. Fresh 100 ugprotein/ml solution of lyophilized dilipidated HMFG (Ceriani et al., Proc. Natl. Acad. Sci.131; 582-586, 1977) in detergent buffer is prepared and sonicated at 10 second intervals fora total of 4 minutes (10 sec. of sonication, followed by a 10 sec. silent period) using adouble step micro tip horn at 25 watts on a Sonifier Cell Disrupter 185 (Branson,Danbury, CT) at 4 ° C. HMFG solutions at concentrations of 0, 10, 33.3, 100, 333.3, and1000 ng protein/ml are prepared in spun female sera, and 3 aliquots of 180 pl of eachHMFG level in normal female sera are pipetted into 400 pl polyethylene microcentrifugetubes (West Coast Sci. Emeryville, CA). 150 ul of 6.6% PEG solution is added to eachmicrocentrifuge tube, and the tubes are incubated overnight on a rotating shaker at roomtemperature.Test samples are processed in a comparable manner, by centrifuging 300-350 ul ofthe eluted nitrocellulose filtrate in solution (or, alternatively, of mammary fluid or otherassay sample alternative) in a 400 pl microcentrifuge tube for 5-7 min. at 10,240 rpm at10° C. The microcentrifuge tubes are then cut with a razor blade below the white bandformed by the sera (if there was one) and 180 ul of remaining sera is transferred to a newmicrocentrifuge tube. 150 pl of a 6.6% PEG solution is then added to eachmicrocentrifuge tube, and the tubes are incubated overnight on a rotating shaker at roomtemperature.Day two(I) 0t—HME is diluted to its appropriate concentration in detergent buffer. Theantibody solution has stoichiometric or lesser amounts of a-HME to 6 ng HMFG protein1015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/ 1486327equivalent (prot. eq.). Six ng of HMFG is covalently bound to microtiter plates by themethylated BSA procedure previously described by Ceriani, In: Kennet et al., (eds)Monoclonal Antibodies and Functional Cell Lines, Plenum Press, New York, 1984, pp.398-402, incorporated herein by reference in its entirety.(2) To process test samples and control samples on the second day,microcentrifuge tubes are centrifuged for 7 min. at 10,240 rpm at 10 ° C in a SHMT rotorwith a Sorvall RCSC centrifuge. In triplicate, 55 ul of supernatant is pipetted into emptymicrotiter plate wells (Dynatech, Alexandria, VA), and any precipitate pelleted is leftundisturbed. 25 [ll of 6.6% PEG solution is added to each well. 30 it! of ot-HME dilutedin detergent buffer is also added to each well, and a non-porous Scotch® tape is placedover the wells to avoid evaporation. The microtiter plate is then incubated overnight atroom temperature on a rotary shaker.Day Three(1) The microtiter plates are centrifuged (3000 r.p.m.) for 30 minutes at roomtemperature to decant suspended perceptible matter.(2) 50 pl of RIA buffer is added to wells of microtiter plates containing 6 ngHMFG and aspirated off after 5 minutes.(3) The total contents of microtiter plates from I), save for any precipitationinduced by the PEG and already pelleted, are carefully transferred to the wellsof another set of microtiter plates containing 6 ng HMFG per well (Day 2,1),above.(4) The microtiter plates are incubated for 3 hours with rotating agitation at roomtemperature.(5) The plates are washed 5 times with RIA buffer using Dynadrop SR-1automatic dispenser form Dynatech.(6) 50 ttl of the radioiodinated affinity—purified rabbit anti-mouse immunoglobulindiluted in RIA buffer is then adder per well.(7) The plate is covered with tape and incubated with rotating agitation for 2 hoursat room temperature.(8) The plate is washed 5 times with RIA buffer.(9) Wells are cut and counted in a gamma counter.15202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486328The results of these assays will yield important information concerning thepresence and/or status of cancer in patients, comparable in scope and v.alue to the dataprovided by serum assays conducted for the HME-Ag breast cancer marker by Ceriani etal., Breast Cancer Res. Treat. 1;: 161-174, 1990. By selecting patient and controlsamples and developing and evaluating comparative data according to the proceduresfollowed by Ceriani and his coworkers, the assay methods of the invention will also bereadily adapted for use in direct clinical applications to determine both prognostic andtreatment related variables in breast cancer patients. Reagents and conditions for theassays can of course be substituted or adjusted depending on a variety of anticipatedvariables, by applying well known immunological methods and principles.EXAMPLE 5Competitive Radioimmunoassav for Non-Penetrating Glvcoprotein in Biological SamplesFrom Mammagy FluidThis competitive radioimmunoassay is based on the displacement by breastepithelial antigens contained in biological samples from mammary fluid obtainedaccording to the methods of the invention of the binding of stoichiometric or lesserquantities of the monoclonal antibody Mc5 to a solid-phase—bound antigen known as non-penetrating glycoprotein (NPGP) contained in HMFG. HMFG is bound to a solid supportand exposed to the Mc5 antibody during an incubation period allowing the antibody tobind the NPGP antigen in solid phase-bound HMFG. The presence and/or level of NPGPin the biological sample is ultimately examined by ability of the sample to compete forMc5 binding to the NPGP antigen in the solid phase-bound HMFG, as detected and/ormeasured using a radiolabeled goat anti-mouse antibody to bind and label the Mc5antibody probe.Buffer and other solutions and reagents in this example are generally the same asthose used for the HME-Ags polyclonal antibody radioimmunoassay described inExample 4, above. To provide test samples for the assay, biological samples frommammary fluid contained on nitrocellulose filters are eluted electrophoretically intophosphate buffered saline, according to standard methods. Alternatively, wholemammary fluid or other types of biological samples obtained from mammary fluid can beconstituted in an appropriate medium or mixture to provide a test sample for the assay.1015202530CA 02264277 1999-02-25wo 98/08976 PCT/US97/ 1486329Once the test sample is thus provided, it is then assayed according to the NPGP/Mc5radioimmunoassay (RIA) methods described in Ceriani et 211., Breast Cancer Res. Treat._l_5_: l6l-174, 1990 (incorporated herein by reference in its entirety), as follows:I) 400 pl of pooled normal female mammary fluid (exposed to nitrocellulosefilters and eluted in the same manner as the nitrocellulose adsorbed, eluted testsample, or alternatively provided as normal whole mammary fluid or othertypes of biological samples obtained from normal mammary fluid constitutedin an appropriate medium or mixture to provide a test sample) to provide asuitable control sample, which is diluted to 2.4 ml using RIA buffer at a 1:6concentration.2) A 500 pg/ml solution of lypholized HMFG is prepared in 1X PBS with 0.3%Triton-X-100, 0.05% sodium azide, and sonicated using a double step microtip horn at 25 watts on a Sonifier Cell Disrupter 185 (Branson, Danbury, CT)for 4 minutes ( 10 sec. sonication, 10 sec. silent period, at 4 ’ C).3) Solutions to prepare a standard curve are prepared using the 2.4 ml 1:6 normalfemale serum and increasing amounts of HMFG (0, 0.25, 2.5, 25, 50 pg/mlHMFG, as described above in Example 4).4) Each test assay sample is diluted 1:6 with RIA buffer (40 pl of serum to 200 plRIA buffer) to form a diluted test assay sample, and vortexed.5) Mc5 stock solution is prepared so that it contains less than stoichiometricamounts of antibody to l00 ng protein/well of HMFG covalently bound tomicrotiter plates prepared as previously described by Ceriani. In: Kennet et al.,(eds) Monoclonal Antibodies and Functional Cell Lines, Plenum Press, NewYork, 1984, pp. 398-402, incorporated herein by reference in its entirety6) 200 pl RIA buffer are added to each well of 100 ng HMFG and then aspiratedafter 5 minutes.7) To prepare a standard curve, 30 pl of HMFG standardizing solutions (as in 3above) are added in quadruplicate to a 100 ng protein/well HMFG microtiterwell.8) 30 pl of diluted test assay sample (or, alternatively, of mammary fluid or otherassay sample alternative) are added in triplicate to 100 ng/well HMFGmicrotiter wells.101520CA 02264277 1999-02-25WO 98/08976 PCT/US97/14863309) To each well 20 pl of the Mc5 stock solution is added.10) Microtiter plates are covered with nonporous Scotch® tape and incubatedovernight at room temperature on a rotating agitator.l 1) The next day the wells are aspirated and washed 5 times with RIA buffer.12) To each well 50 in of 200,000 cprnl 50 ul '“I—goat anti-mouse antibody aredispensed. The wells are covered with nonporous tape and placed on arotating agitator for 3 hours at room temperature.13) Wells are washed 5 times with RIA buffer.14) Each well is cut and the radioactivity is counted using a gamma counter.The results of these assays will yield important information concerning thepresence and/or status of cancer in patients, comparable in scope and value to the dataprovided by serum assays conducted for the NPGP breast cancer marker by Ceriani et al.,Breast Cancer Res. Treat. 1_§: 161-174, 1990. By selecting patient and control samplesand developing and evaluating data according to the well known procedures followed byCeriani and his coworkers, the assay methods of the invention will be readily adapted foruse in direct clinical applications to determine both prognostic and treatment relatedvariables in breast cancer patients. As will be understood by those skilled in the art,reagents and conditions for the assays can be substituted or adjusted depending on avariety of anticipated variables, according to well known immunological methods andprinciples.I015202530W0 98l08976CA 02264277 1999-02-25PCT/US97/ 148633 IEXAMPLE 6Solid Phase Immunoassay for Mucinous Carcinoma Associated Antigen in MammaryE!This example uses a sensitive, solid phase immunoassay to detect the mucinouscarcinoma associated antigen (MCA) in biological samples from mammary fluid obtainedaccording to the methods of the invention. MCA concentrations are detennined using anantibody-bead immunoassay kit provided by Hoffman—La Roche (Basel, Switzerland), andusing the reagents and procedures provided by the manufacturer and described in furtherdetail in Eskelinen et al., Anticancer Res. 2: 437-440, 1989. Briefly, test assay samples ofwhole mammary fluid and standards are first incubated with MCA monoclonal antibodybeads and then, after appropriate washings, enzyme (horseradish peroxidase) labeledsecondary antibody is added. During the second incubation the anti—MCA enzymeconjugates are attached to the antibody antigen complex on the beads. Excess conjugatesare removed by washing and, finally, enzyme substrate are added and the color formed isrecorded.The solid phase assay format presented in this example can be adapted for use in awide array of other assays to detect and/or measure other cancer markers besides theMCA marker, with enhanced sensitivity. In addition, the results of these assays can beevaluated along with those of complementary assays detecting and/or measuring differentmarkers to yield more precise information concerning the presence and/or status of cancerin patients, as exemplified by the combinatorial MCA/CA l5—3 assays described byEskelinen et al., Anticancer Res. 2: 437-440, 1989; see also Eskelinen et al., AnticancerRes. §: 665-668, 1988, each incorporated herein by reference in its entirety.EXAMPLE 7Western Analysis of Proteins From Cellular Fractions of Human Mammary Fluid UsingPolyclonal and Monoclonal Antibody Probes to Detect VasopressinA variety of assays are provided by the present invention that focus on cellularsamples from human mammary fluid. In general, these assays rely on isolation bystandard separation methods (eg. centrifugation, sucrose gradient, etc.) of cells,membranes or other cell components from whole mammary fluid expressed according tothe above methods. Biological samples containing whole cells from expressed mammary M,“,~w_‘__,_“___1N_u_,,,,,_‘W___U,_,__,____,,,_‘,_o,,,_,¢_,,___,_,_,,,,,,, , . .. . . . 2 . ..,. r,....~....i.....r......,.....n.....i.....«m...............i......... ., 15202530WO 98108976CA 02264277 1999-02-25PCT/US97/1486332fluid are particularly useful for cytological and cytochemical examination to detect andevaluate breast cancer in patients. Biological samples containing purified cell membranefractions from human mammary fluid are particularly useful in this context, for exampleto detect and/or measure breast cancer markers that are expressed by alveolar-ductal cellsas intracellular or membrane bound proteins and are therefore not as readily detected inliquid fractions of mammary fluid as secreted proteins.The present example focuses on assays for detecting the peptide hormonevasopressin in biological samples from mammary fluid, using methods adapted fromNorth et al., Breast Cancer Res. Treat. §_{1_: 229-235, 1995. Specifically, this assay uses atest sample of crude protein isolated from a pooled sample of cells obtained fromexpressed mammary fluid. The cells are separated from whole mammary fluid accordingto standard methods, and crude protein is extracted from the cells by sonication in 100volumes of 0.1 M HC 1. The resulting protein suspensions are then centrifuged at 1500 xg for 10 min. at ambient temperature, and soluble protein is precipitated with 40% TCA.This protein is pelleted by centrifugation at 10,000 x g for 2 min. TCA is then removedfrom pellets by washing (x 2) with ether. Protein is resuspended in 0.1 M Tris HCl(pH8.7), reduced with mercaptoethanol at l00° C for 5 min. (and in some cases S-alkylated with N-ethyl maleimide), and subjected to SDS-PAGE electrophoresis on 15%gels at pH 9.3 using the method of Laemeli, J 22.1: 680-685, I970, incorporatedherein by reference in its entirety. Separated proteins are then electrophoreticallytransferred with 20 mM Tris glycine (pH 8.0) to Immobilon PVDF membranes (Millipore,Bedford, MA). These membranes are blocked with a 5% non-fat milk solution, washed (1x 15 min., 2 x 5 min.) with PBS containing 0.5% Triton, and incubated with preparationsof mouse monoclonal antibody to VP-HNP, with rabbit polyclonal antibodies to VP, withrabbit polyclonal antibodies to VAG, or with ubiquitous mouse or rabbit IgG (negativecontrols) (for description of antibodies and antibody preparation see North et al., BristCancer Res. Treat. fir 229-235, 1995, incorporated herein by reference in its entirely), forl h at ambient temperature. Following a second wash in PBS-Triton (l x 15 minnn., 2 x 5min.), the membranes are treated, respectively, with goat anti-mouse IgG-horseradishperoxidase conjugate or goat anti—rabbit IgG-horseradish peroxidase conjugate for l h,and then washed with PBS—Triton (1 x l5 min., 4 x 5 min.). Immunoreactive proteins arevisualized using an ECL Western Blotting Detection System with exposure of x—ray filml015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/148633 3from 10 seconds to 5 min. Prestained SDS-PAGE standard proteins are employed asmolecular size markers.Recent studies suggest that vasopressin is universally expressed in breastcarcinoma and is absent from normal breast cells. North et a1., Breast Cancer Res. Treat.3: 229-235, 1995. These and other results indicate that vasopressin and its relatives areimportant breast cancer markers which can be readily detected using immunologicalassays of proteins isolated from breast tumor cells. Accordingly, the results of the presentexample using cell samples isolated from human mammary fluid are also expected toyield important information concerning the presence and/or status of cancer in patients.EXAMPLE 8Quantification of Carcinoembrvonic Antigen in Biological Samples From MammaryFluid by Dot Immunoblotting AssayAmong the more sensitive assays of the invention, useful for measuring low levelsof breast cancer markers and for detecting markers when only small volumes of expressedmammary fluid are available, is the dot immunoblotting assay. In the present example,carcinoembryonic antigen (CEA) is measured in whole mammary fluid using an Elmotechanti-CEA monoclonal antibody kit (Mochid Pharmaceutical C0,, Tokyo, Japan) in a dotblot assay format. Briefly, anti—CEA monoclonal antibody is diluted to appropriateconcentrations and coated on the plastic film. Aliquots (5 ill) of either standard CEAsolution (0, I00, 200, and 500 ng/ml), or of the whole mammary fluid assay sample, aresmeared on the immobilized film. Assay standards are prepared from purified antigenpreparations, in accordance with the Elmotec kit manufacturer's instructions. Ifnecessary, 1000 ng/ml CEA solution is also used as a standard. After drying at roomtemperature, the film is exposed to peroxidase—conjugated anti—CEA antibody for 20 minat room temperature. The film is then washed extensively with l M saline containing0.5% (v/v) Tween 20. The enzyme reaction is visualized using tetramethylbenzidine as achromogen. The developing solution consists of 0.05 mM tetramethylbenzidine and0.01% hydrogen peroxide in Mcllvain buffer (0.1% M phosphate—citrate buffer), pH 5.0,containing 10% methanol. The concentration of CEA in the mammary fluid assay sampleis determined by comparing the color intensities with a corresponding standard.I015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486334The assay disclosed in the present example, and related assays incorporatingantibodies to other tumor markers besides CEA, are particularly useful for measuring lowlevels of breast cancer markers and for detecting markers in limited sample volumes. Theresults of these assays will yield important information to determine both prognostic andtreatment related variables in breast cancer patients. As will be understood by thoseskilled in the art, reagents and conditions for the assays can be substituted or adjusteddepending on a variety of anticipated variables, according to well known immunologicalmethods and principles.EXAMPLE 9Detection of Procathepsin D and Cathepsin D Activity in Biological Samples FromMammary FluidCathepsin D is a lysosomal aspartic proteinase which has been studied intensivelyas a marker for cancer processes necessary for metastasis. In the present example,polyclonal antibodies against procathepsin D are used to immunoprecipitate andimmunochemically detect proteins from whole mammary fluid or cell lysates frommammary fluid, generally according to the methods disclosed in Vetvicka et al., Biochem.Mol. Biol. Int’l. Q: 921-928, 1993 (incorporated herein by reference in its entirety).Alternatively, or as a complementary assay, the protease activity of cathepsin D isdetected, also according to the methods disclosed in Vetvicka et al. (Id.). Briefly, pooledwhole mammary fluid (preferably 3 ml if available) is diluted with 3 ml of buffer A (50mM Tris.HCl, 5 mM CaCl2, 1 mM MgCl2, 500 mM NaCl pH 7.2). The suspension iscentrifuged for 30 minutes at 10,000 g. The resulting water phase is centrifuged againunder the same conditions. The soluble part (total of approximately 4.5 ml) is loaded on a1 ml column of Concanavalin A Sepharose (Pharmacia, Uppsala, Sweden) equilibrated inbuffer A, and after washing with buffer A the retained proteins are eluted using 0.75 Mmethyl a—D-mannopyranoside. The fractions (250 pl) are analyzed for cathepsin Dactivity using the MC hemoglobin assay as described by Lin et al., J. Biol. Chem. 2_6_4:4482-4489, 1989 (incorporated herein by reference in its entirety), by western blots and bysilver—stained electrophoresis. The inhibition of human milk procathepsin D isaccomplished by adding 2 ul of 1 mM pepstatin A (Boehringer Manheim, Germany)dissolved in methanol to the reaction mixture.1015202530WO 98/08976CA 02264277 1999-02-25PCT/US97/1486335This assay provides but one example of many possible embodiments of theinvention that incorporate known biochemical assays, in addition to, or supplemental toimmunological assays, to evaluate biological samples from mammary fluid to determinecancer related variables. The fundamental methods provided herein for obtaining samplesfrom human mammary fluid render these assays readily adaptable for widespread clinicalapplication to detect and/or measure the activity of a subject breast cancer marker within anon—invasive screening protocol.Those with ordinary skill in the art will appreciate that other embodiments andvariations of the invention are possible which employ the same inventive conceptsdescribed above. Most particularly, a wide and rapidly expanding array of useful breastcancer markers (including proteins, DNA and RNA sequences and other markers) andprobes (including immunological, nucleotide and biochemical probes) are readilyavailable for adaptation and use within the methods and kits of the invention. Thesemarkers and probes are described or referenced to a large extent in the literature cited andincorporated within the present disclosure, or are elsewhere published in the literature orwell known in the art. Among these known and emerging markers and probes, usefulexamples within the invention include Her 2 (also known as erbB-2 and neu). Her 2 is atransmembrane glycoprotein growth factor receptor of the EGF receptor family encodedby a gene located on chromosome l7q, a region of frequent amplification in breast cancercell lines. This marker is highly predictive of breast cancer and can be detected in cellularsamples of the invention using known nucleotide probes to detect genetic defects in Her 2,or to detect and/or measure mRNA to determine overexpression of Her 2 linked toincreased proliferation of cancer cells. (See for example, Visscher et al., In Weinstein andGraham (eds) Advances in Pathology and Laboratory Medicine, vol 5, St Louis, MosbyYuear Book, l992, pp. 123-161; Barbareschi et al., Am. J. Clin. Pathol. %: 408-418,1992; Slamon et al., §_c_igr_ig§ 2_3_§: 177-182, 1987; each incorporated herein by reference inits entirety). Protein levels of Her 2 are also readily detected using availableimmunological probes. (For review see Porter-Jordan et al., Hematol. Oncol. Clin. NorthAmer. §: 73-100, 1994 and articles cited on page 80 therein. each incorporated herein byreference in its entirety). Additional markers for use within the invention include EGFand the EGF receptor, for which immunological and non-immunological probes and assaymethods readily adaptable within the invention are characterized in detail at page 80-81 of l015WO 98/08976CA 02264277 1999-02-25PCT /U S97/1486336Porter—Jordan et al., Hematol. Oncol. Clin. North Amer. §: 73-100, 1994 and in thereferences cited therein, each incorporated herein by reference in its entirety. Additionalexamples of proliferation markers, growth factors and receptors, proteases, adhesionfactors, angiogenic factors, oncogenes and tumor suppressor genes that may provideuseful breast cancer markers and probes within the methods and kits of the inventioninclude Ki67 Growth Factor, Cyclin D1, Proliferating Cell Nuclear Antigen,Transforming Growth Factor (1, Tissue Plasminogen Activator, Insulin Growth FactorReceptors, Collagenase Type IV, Laminin Receptor, Integrins, p53, rb, nm23, ras, c—myc,Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, and many others, the majority of which are described along withavailable immunological and non—immunological probes and assay methods readilyadaptable within the invention in the review by Porter-Jordan et al., Hematol. Oncol. Clin.North Amer. §: 73-100, 1994 and in the references cited therein, each incorporated hereinby reference in its entirety. Adaptation of these markers and probes within the inventioncan be readily accomplished based on the teachings provided herein and in the citedreferences, supplemented by general knowledge in the art. Therefore, the invention is notto be limited by the above description, but is to be determined in scope by the claimswhich follow.

Claims (47)

WHAT IS CLAIMED IS:

1. ~A sample collection device for collecting a biological sample from a mammary organ of a subject, comprising:
a breast engaging member for application to a breast of the subject to establish fluid connection between the engaging member and a nipple or alveolar duct of the breast;
means for applying negative pressure connected with said breast engaging member for applying negative pressure at a surface of the nipple or within an alveolar duct of the breast to induce or facilitate breast fluid expression from the nipple or alveolar duct;
and a solid phase sample collection medium in fluid connection with said breast engaging member for receiving a sample of expressed breast fluid from the nipple or alveolar duct.

2. ~The sample collection device of claim 1, wherein said pressure changing means comprises a vacuum pump in gaseous connection with said breast engaging member that exerts negative pressure on the nipple surface or within the alveolar duct.

3. ~The sample collection device of claim I or 2, wherein said solid phase sample collection medium in fluid connection with said breast engaging member selectively adsorbs, binds filters, or otherwise processes desired components of the mammary fluid for incorporation into an assay for detecting a breast disease marker.

4. ~The sample collection device of any one of claims 1 to 3, wherein said solid phase sample collection medium is in fluid contact with said nipple or alveolar duct when the breast engaging member is applied to the breast and positive or negative pressure is generated by said negative pressure changing means.

5. ~The sample collection device of any one of claims 1 to 4, wherein said solid phase sample collection medium is placed in fluid connection with a breast pump.

6. ~The sample collection device of any one of claims 1 to 5, wherein said solid phase sample collection medium is selected from the group consisting of microscopic glass slides, nitrocellulose filter, affinity column, dot blot matrix, adsorbant media, binding media and filtering media.

7. The sample collection device of any one of claims 1 to 5, wherein said solid phase sample collection medium comprises a filter or membrane.

8. ~The sample collection device of any one of claims 1 to 5, wherein said solid phase sample collection medium comprises a nitrocellulose filter or membrane.

9. ~The sample collection device of any one of claims 1 to5, wherein said solid phase sample collection medium functions to receive or partition cells, cellular constituents, purified or bulk proteins, glycoproteins, peptides, or nucleotides from the mammary fluid.

10. ~The sample collection device of any one of claims 1 to 9, wherein said biological sample is selected from the group consisting of whole mammary fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, and nucleotide components of mammary fluid.

11. ~The sample collection device of any one of claims 1 to 10, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of a protein, a peptide, a glycoprotein, a lipid, a glycolipid, a DNA
polynucleotide, an RNA polynucleotide, and a combination thereof.

12. ~The sample collection device of any one of claims 1 to 11, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of Ki67 Growth Factor, Cyclins, Proliferating Cell Nuclear Antigen, Estrogen, Epidermal Growth Factor, Transforming Growth Factor a, Tissue Plasminogen Activators, Insulin Growth Factor Receptors, Epidermal Growth Factor Receptors, Collagenases, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, CEA, HME
antigens, MCA, PSA, Vasopressin, and Cathepsin D, and a combination thereof.

13. ~A non-invasive method for obtaining a biological sample from a mammary organ of a subject, comprising the steps of:
applying a sample collection device for collecting said biological sample from said subject comprising:
a breast engaging member for application to a breast of the subject to establish fluid connection between the engaging member and a nipple or alveolar duct of the breast;
means for applying negative pressure connected with said breast engaging member for applying negative pressure at a surface of the nipple or within an alveolar duct of the breast to induce or facilitate breast fluid expression from the nipple or alveolar duct; and a solid phase sample collection medium in fluid connection with said breast engaging member for receiving a sample of expressed breast fluid from the nipple or alveolar duct; and, concurrent or subsequent to expression of said breast fluid, collecting the biological sample comprising the expressed mammary fluid or a component thereof that includes one or more breast disease markers on or within the solid phase sample collection medium.

14. ~The method for obtaining the biological sample according to claim 13, wherein said pressure changing means comprises a vacuum pump in gaseous connection with said breast engaging member or alveolar duct that exerts negative pressure on an exterior of the nipple or within the alveolar duct.

15. ~The method for obtaining the biological sample according to claim 13 or 14, wherein said solid phase sample collection medium is in fluid contact with said nipple or alveolar duct when the breast engaging member is applied to the breast and positive or negative pressure is generated by said negative pressure changing means.

16. The method for obtaining the biological sample according to any one of claims 13 to 15, wherein said solid phase sample collection medium is placed in fluid connection with a breast pump.

17. The method for obtaining the biological sample according to any one of claims 13 to 16, wherein said solid phase sample collection medium is selected from the group consisting of microscopic glass slides, nitrocellulose filter, affinity column, dot blot matrix, adsorbant media, binding media and filtering media.

18. The method for obtaining the biological sample according to any one of claims 13 to 16, wherein said solid phase sample collection medium comprises a filter or membrane.

19. The method for obtaining the biological sample according to any one of claims 13 to 16, wherein said solid phase sample collection medium comprises a nitrocellulose filter or membrane.

20. The method for obtaining the biological sample according to any one of claims 13 to 19, wherein said solid phase sample collection medium functions to receive or partition cells, cellular constituents, purified or bulk proteins, glycoproteins, peptides, or nucleotides from the mammary fluid.

21. The method for obtaining the biological sample according to any one of claims 13 to 20, wherein said biological sample is selected from the group consisting of whole mammary fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, and nucleotide components of mammary fluid.

22. The method for obtaining the biological sample according to any one of claims 13 to 21, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of a protein, a peptide, a glycoprotein, a lipid, a glycolipid, a DNA polynucleotide, an RNA polynucleotide, and a combination thereof.

23. The method for obtaining the biological sample according to any one of claims 13 to 21, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of Ki67 Growth Factor, Cyclins, Proliferating Cell Nuclear Antigen, Estrogen, Epidermal Growth Factor, Transforming Growth Factor a, Tissue Plasminogen Activators, Insulin Growth Factor Receptors, Epidermal Growth Factor Receptors, Collagenases, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, CEA, HME antigens, MCA, PSA, Vasopressin, and Cathepsin D, and a combination thereof.

24. The method for obtaining the biological sample according to any one of claims 13 to 23, wherein said step of collecting said biological sample includes contacting the expressed mammary fluid to the solid phase sample collection medium that retains at least one or more breast disease markers from the expressed mammary fluid.

25. An assay method for determining the presence or amount of one or more breast disease markers in a biological sample obtained from a mammary organ of a patient, comprising the steps of:
applying a sample collection device for collecting said biological sample from said subject comprising:
a breast engaging member for application to a breast of the subject to establish fluid connection between the engaging member and a nipple or alveolar duct of the breast;
means for applying negative pressure connected with said breast engaging member for applying negative pressure at a surface of the nipple or within an alveolar duct of the breast to induce or facilitate breast fluid expression from the nipple or alveolar duct; and a solid phase sample collection medium in fluid connection with said breast engaging member for receiving a sample of expressed breast fluid from the nipple or alveolar duct; and, concurrent or subsequent to expression of said breast fluid, collecting the biological sample comprising the expressed mammary fluid or a component thereof that includes one or more breast disease markers on or within the solid phase sample collection medium; and detecting the presence or amount of said one or more breast disease markers in the sample.

26. The assay method according to claim 25, wherein said pressure changing means comprises a vacuum pump in gaseous connection with said breast engaging member or alveolar duct that exerts negative pressure on an exterior of the nipple or within the alveolar duct.

27. The assay method according to claim 25 or 26, wherein said solid phase sample collection medium is in fluid contact with said nipple or alveolar duct when the breast engaging member is applied to the breast and positive or negative pressure is generated by said negative pressure changing means.

28. The assay method according to any one of claims 25 to 27, wherein said solid phase sample collection medium is placed in fluid connection with a breast pump.

29. The assay method according to any one of claims 25 to 28, wherein said solid phase sample collection medium is selected from the group consisting of microscopic glass slides, nitrocellulose filter, affinity column, dot blot matrix, adsorbant media, binding media and filtering media.

30. The assay method according to any one of claims 25 to 28, wherein said solid phase sample collection medium comprises a filter or membrane.

31. The assay method according to any one of claims 25 to 28, wherein said solid phase sample collection medium comprises a nitrocellulose filter or membrane.

32. The assay method according to any one of claims 25 to 31, wherein said solid phase sample collection medium functions to receive or partition cells, cellular constituents, purified or bulk proteins, glycoproteins, peptides, or nucleotides from the mammary fluid.

33. The assay method according to claim any one of claims 25 to 32 wherein said biological sample is selected from the group consisting of whole mammary fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, and nucleotide components of mammary fluid.

34, The assay method according to any one of claims 25 to 33, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of a protein, a peptide, a glycoprotein, a lipid, a glycolipid, a DNA
polynucleotide, an RNA polynucleotide, and a combination thereof.

35. The assay method according to any one of claims 25 to 34, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of Ki67 Growth Factor, Cyclins, Proliferating Cell Nuclear Antigen, Estrogen, Epidermal Growth Factor, Transforming Growth Factor a, Tissue Plasminogen Activators, Insulin Growth Factor Receptors, Epidermal Growth Factor Receptors, Collagenases, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actm, CEA, HME

antigens, MCA, PSA, Vasopressin, and Cathepsin D, and a combination thereof.

36. The assay method according to any one of claims 25 to 35, wherein said step of collecting said biological sample includes contacting the expressed mammary fluid to the solid phase sample collection medium that retains at least one or more breast disease markers from the expressed mammary fluid.

37. A non-invasive method for obtaining a biological sample from a mammary organ of a subject, comprising the steps of:
applying negative pressure to a breast, nipple or alveolar duct of the subject to induce or facilitate breast fluid expression from the nipple or alveolar duct;
applying a breast engaging member to the breast of the subject to establish a fluid connection between the nipple or alveolar duct and the breast engaging member;

providing a solid phase sample collection medium in fluid connection with said breast engaging member for receiving a sample of expressed breast fluid from the nipple or alveolar duct; and concurrent or subsequent to expression of said breast fluid, collecting the biological sample comprising the expressed mammary fluid or a component thereof that includes one or more breast disease markers on or within the solid phase sample collection medium.

38. The method for obtaining the biological sample of claim 37, wherein the step of applying positive or negative pressure to the breast, nipple or alveolar duct comprises activating a vacuum pump in gaseous connection with said breast engaging member to exert negative pressure on the nipple and/or the alveolar duct.

39. The method for obtaining the biological sample of claim 37 or 38, wherein said solid phase sample collection medium is in fluid contact with said nipple or alveolar duct when the breast engaging member is applied to the breast and positive or negative pressure is generated by said negative pressure changing means.

40. The method for obtaining the biological sample of any one of claims 37 to 39, wherein said solid phase sample collection medium is placed in fluid connection with a breast pump.

41. The method for obtaining the biological sample of any one of claims 37 to 40, wherein said solid phase sample collection medium is selected from the group consisting of microscopic glass slides, nitrocellulose filter, affinity column, dot blot matrix, adsorbant media, binding media and filtering media.

42. The method for obtaining the biological sample of any one of claims 37 to 40, wherein said solid phase sample collection medium comprises a filter or membrane.

43. The method for obtaining the biological sample of any one of claims 37 to 40, wherein said solid phase sample collection medium comprises a nitrocellulose filter or membrane.

44. The method for obtaining the biological sample of any one of claims 37 to 43, wherein said biological sample is selected from the group consisting of whole mammary fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, and nucleotide components of mammary fluid.

45. The method for obtaining the biological sample of any one of claims 37 to 44, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of a protein, a peptide, a glycoprotein, a lipid, a glycolipid, a DNA polynucleotide, an RNA polynucleotide, and a combination thereof.

46. The method for obtaining the biological sample of any one of claims 37 to 45, wherein said biological sample comprises one or more breast disease markers selected from the group consisting of Ki67 Growth Factor, Cyclins, Proliferating Cell Nuclear Antigen, Estrogen, Epidermal Growth Factor, Transforming Growth Factor a, Tissue Plasminogen Activators, Insulin Growth Factor Receptors, Epidermal Growth Factor Receptors, Collagenases, Laiminin Receptor, Integrins, p53, rb, nm23, ras, c-myc, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, CEA, HME antigens, MCA, PSA, Vasopressin, and Cathepsin D, and a combination thereof

47. The method for obtaining the biological sample of any one of claims 37 to 46, wherein said step of collecting said biological sample includes contacting the expressed mammary fluid to the solid phase sample collection medium that retains at least one or more breast disease markers from the expressed mammary fluid.