JP2004073880A - Device for collecting mamma fluid specimen of breast disease including cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable to collect a biological specimen from a patient's mamma organ. <P>SOLUTION: A specimen collecting device is constituted to collect the biological specimen from the patient' mamma organ. The specimen collecting device contains a mamma conformity member for applying to the patient's mamma for establishing fluid connection between the mamma conformity member and the mamma nipple or a duct in the lobule; a pressure changing means connected to the mamma conformity member for changing the pressure on the surface of the mamma nipple or in the duct in the lobule for inducing or promoting the generation of the mamma fluid from the nipple or the duct in the lobule; and a solid state specimen collecting medium which is present for fluid-connecting with the mamma conformity member for receiving the mamma fluid specimen generated from the nipple or the duct in the lobule. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to methods and kits for collecting and assaying biological samples from breast fluid. More particularly, the present invention provides for collecting and assaying fluid and cytological samples from the mammary gland of a mammalian subject to evaluate breast disease including infection, precancerous symptoms, susceptibility of cancer and cancer, Methods and kits for diagnosing and managing.

Breast cancer is the most common form of cancer in women and is the second leading cause of cancer death in humans. Despite the many recent developments in breast cancer diagnosis and treatment, the rise of the disease has been steadily increasing since 1940 at a rate of about 1% / year. Today, women living in North America have one in eight chances of developing breast cancer during their lifetime.

The current widespread use of mammography has improved the detection of breast cancer. Nevertheless, the mortality rate for breast cancer remains unchanged at about 27 per 100,000 women. Often, breast cancer is found at an advanced stage, so therapeutic options and survival are very limited. Therefore, more sensitive and reliable methods are needed to detect small (less than 2 cm in diameter), early stage intraepithelial neoplasia of the breast. Such a method would significantly improve breast cancer survival, as suggested by the successful use of Papinicolou smears for early detection and treatment of cervical cancer.

Significant in distinguishing malignant and benign breast disease, staging of known breast cancer, and identifying different types of breast cancer (eg, estrogen-dependent / non-oestrogen-dependent tumors), in addition to the problem of early detection Problems remain. Recent efforts to develop improved methods of breast cancer detection, staging and classification have focused on promising sequences of so-called cancer "markers". A cancer marker is typically expressed specifically by a cancer cell (eg, as a cell surface or as a secreted protein), or is measurably increased or decreased by a cancer cell relative to a normal cell. It is a protein expressed at a defined level. Other cancer markers can include specific DNA or RNA sequences that mark a detrimental change or change in the pattern or level of expression of a gene associated with a particular form of cancer.

A number of different breast cancer markers have been identified to date and these have been shown to have significant value in determining prognostic and / or treatment-related variables. Prognostic variables are variables that serve to predict the outcome of a disease, for example, the likelihood of relapse or survival. Treatment-related variables are those that predict the likelihood of success or failure of a given treatment plan. Certain breast cancer markers clearly serve both functions. For example, estrogen receptor levels are predictive of recurrence and survival in breast cancer patients and independent of response to endocrine therapy, independent of treatment. Pertschuk et al., Cancer, 66: 1663-1670, 1990; Parl and Posey, Hum.Pathol., 19: 960-966, 1988; Kinsel et al., Cancer Res., 49: 1052-1056, 1989; Anderson And Poulson, Cancer, 65: 1901-1980, 1989.

Utilizing specific breast cancer markers for screening and diagnostic, staging and classification, monitoring and / or therapeutic purposes depends on the nature and activity of the marker in question. For a general overview of breast cancer markers, see: Portr-Jordan et al., Hemotol.Oncol.Clin.North Amer., 8: 73-100, 1994; and Greiner, Pharmaceutical Tech., May. 1993, pp.28-44. As reflected in these overviews, the primary focus for the development of breast cancer markers has focused on the overlapping areas of tumor development, tumor growth and cancer invasion. Tumor development and tumor growth can be assessed using a variety of cell proliferation markers, such as Ki67, cyclin D1, and proliferating cell nuclear antigen (PCNA), some of which are also important oncogenes. There may be.

Tumor growth can also be assessed using various growth factor and hormonal markers (eg, estrogen, epidermal growth factor (EGF), erB-2, transforming growth factor (TGF) α), May be over-expressed, under-expressed, or show altered activity in cancer cells. Similarly, receptors for autocrine or exocrine growth factor and hormones (eg, insulin growth factor (IGF) receptor and EGF receptor) may also show altered expression or activity associated with tumor growth. Finally, tumor growth is supported by angiogenesis, including the elaboration or growth of new blood vessels, and the concomitant expression of angiogenic factors that can serve as markers of tumor development and growth.

In addition to markers of tumor development, growth and proliferation, a number of markers have been identified that can serve as indicators of the likelihood of invasion and / or metastasis in a population of cancer cells. These markers generally reflect altered interactions between cancer cells and their surrounding microenvironment. For example, when cancer cells invade or metastasize, detectable changes may occur in the expression or activity of factors of cell attachment or motility, examples of which include cancer markers, cathepsin D, plasminogen activator, collagenase and Includes other factors. In addition, decreased or over-expression of some putative tumor “suppressor” genes (eg, nm23, p53 and rb) increases the likelihood of metastasis or growth, which is indicative of poor disease outcome. Directly associated with loss of regulation.

In summary, among other cancer markers, growth factors, oncogenes, growth factors and growth factor receptors, angiogenesis factors, proteases, adhesion factors and tumor suppressor genes are associated with the risk, presence, condition or future of cancer in patients. Can provide important information about the behavior of Determining the presence or level of expression or activity of one or more of these cancer markers facilitates the differential diagnosis of patients with uncertain clinical abnormalities, for example, by distinguishing malignant abnormalities from benign abnormalities . In addition, in patients with an established malignancy, cancer markers may be effective in predicting the risk of future recurrence or the likelihood of a particular patient responding to a selected course of treatment. Even more specific information can be obtained by analyzing highly specific cancer markers, or combinations of markers, that can predict a patient's response to a particular drug or treatment option.

Methods for detecting and measuring cancer markers have recently been revolutionized by the development of immunological assays, particularly those that utilize the technology of monoclonal antibodies. Previously, many cancer markers could only be detected or measured using conventional biochemical assays. Such biochemical assays generally required large amounts of test samples and were therefore unsuitable for many clinical applications. In contrast, modern immunoassay techniques can detect and measure cancer markers in relatively small samples, especially when using monoclonal antibodies that specifically recognize targeted marker proteins. it can. Thus, it is now routine to assay for the presence or absence, level, or activity of selected cancer markers by immunohistochemically staining breast tissue obtained via conventional biopsy methods. is there.

Due to the highly sensitive nature of immunohistochemical staining, these methods also require a smaller, needle biopsy that requires less invasive sample collection techniques compared to conventional biopsy specimens. It can be successfully used to detect and measure cancer markers in a sample. In addition, other immunological methods have been developed that allow the detection and measurement of cancer markers in non-cellular samples from patients, such as serum and other biological fluids, and are currently being developed in the art. well known. The use of these alternative sample sources substantially reduces morbidity and cost of the assay, as compared to conventional biopsy sample-based approaches, the application of cancer marker assays in early screening, and invasion. Allows for a low risk monitoring program where no invasive biopsy has been applied.

で For the purpose of assessing breast cancer, it is often undesirable to use conventional or needle biopsies for the assay of cancer markers. Because the primary goal of such an assay is to detect the cancer before it has progressed to a palpable or mammographically detectable tumor stage. Prior to this stage, biopsies are generally contraindicated, making initial screening and low risk monitoring techniques using such samples unsustainable. Therefore, there is a general need in the art to obtain a sample for breast cancer marker assay by means of less invasiveness than a biopsy, eg, withdrawal of serum.

Efforts to use serum samples for breast cancer marker assays have had limited success. Primarily because targeted markers are not detectable in serum or tall tel changes in marker levels or activity cannot be monitored in serum. In addition, the presence of breast cancer in serum probably occurs at the time of micrometastasis, reducing the effectiveness of serum assays for detecting pre-metastatic disease. In contrast, the fluid in the mammary gland itself is much higher and even higher than serum, especially in view of the fact that 80% to 90% of all breast cancers occur in the intraductal epithelium of the mammary gland It is expected to contain biologically significant levels of breast cancer markers.

Fluids within the breast duct are secreted by cells surrounding the alveolar-ductal system, or hormones, growth factors and other hormones comparable to those acting on them. It is expected to contain the protein marker assembly and the protein concentration. Similarly, cells and solid cellular debris that can be used in cytological or immunological assays to evaluate intracellular or cell surface markers that may not be detectable in the liquid fraction of breast fluid The fluid is expected to contain.

Previous attempts to develop non-invasive breast cancer markers utilizing breast fluid samples have included studies of breast fluid obtained from patients exhibiting spontaneous nipple discharge. In one of these studies, performed by Inaji et al., Cancer, 60: 3008-3013, 1987, breast cancer was detected using conventional enzyme-linked immunoassays (ELISA) and sandwich-type monoclonal antibody immunoassays. The level of the marker carcinoembryonic antigen (CEA) was measured. These methods have proven successful and reproducible in that the level of CEA in spontaneously excreted breast fluid provides a susceptibility indicator of breast cancer that is not palpable. In a subsequent study by Inaji et al., Jpn. J. Clin. Oncol., 19: 373-379, 1989, the use of a more sensitive dry chemical dot immunobinding assay for CEA determination The results have been extended.

In this latter study, assessed CEA levels were present in 43% of patients tested with palpable breast tumors and in 73% of patients tested with non-palpable tumors Was reported to be. CEA levels in the excreted breast fluid correlated highly with intratumoral CEA levels, indicating that breast cancer cells closely reflected the level of CEA expression in the CEA content of breast fluid. Based on these results, the authors concluded that an immunoassay for CEA in spontaneously excreted breast fluid was effective in screening for nonpalpable breast cancer.

Although assessment of breast fluid has been shown to be an effective method of screening for nonpalpable breast cancer in women who experience spontaneous nipple drainage, this condition is rare, and the method of Inaji et al. It is not possible to apply to the majority of women who are candidates for screening for breast cancer. Furthermore, the first Inaji report cited above determined that certain patients suffering from spontaneous nipple excretion secreted less than 10 μl of mammary fluid, which was the ELISA and sandwich immunoassay used in that study. In order to be at a critically low level. Other antibodies used in assays for other cancer markers are still less sensitive than the anti-CEA antibodies used by Inaji and co-workers, and therefore dry chemical removal of small samples of spontaneously excreted breast fluid. It may not be sufficiently adaptable or sensitive for use even in immunoassays.

In view of the foregoing, biology for use in assessing, diagnosing, and managing breast disease, including cancer, particularly for screening early stage nonpalpable breast tumors There is a great need in the art for more widely applicable, non-invasive methods and materials for obtaining target samples. Highly specific cancer prognosis and / or treatment related information by assessing, diagnosing, and managing breast disease, particularly by detecting or measuring a selected breast cancer marker, or a panel of breast cancer markers And methods and materials that utilize such readily obtained biological samples to diagnose and manage precancerous symptoms, cancer susceptibility, breast infections, and other breast diseases, Required in relation to.

Accordingly, it is an object of the present invention to provide non-invasive methods and kits for obtaining biological samples that can be used in assays to evaluate, diagnose and manage breast disease, especially cancer. Other objects of the invention are assays and kits that are broadly applicable to a wide range of patients and include assays and kits useful for screening early stage, nonpalpable breast tumors In order to achieve the above object.

Yet another object of the present invention is to detect and / or detect a marker of a selected breast disease, eg, a breast cancer marker, or a panel of breast cancer markers, to provide clinicians with information relevant to a particular prognosis and / or treatment. Or, by measuring, to provide a method and kit utilizing said biological sample for assessing, diagnosing and managing breast disease, preferably breast cancer.

The present invention achieves these and other objects and advantages which will become apparent from the description below by providing a non-invasive method of collecting a biological sample from a breast organ of a mammalian patient. I do. In particular, the methods of the present invention comprise administering an amount of oxytocin or an oxytocin analog that is effective to stimulate expression of breast fluid from the nipple of the patient. Oxytocin is preferably administered intranasally and reaches the target alveolar tract tissue of the breast, where oxytocin stimulates contraction of the myoepithelium of the alveolar tract tissue. In addition, intramuscular or intravascular injections of oxytocin can be performed to generate a myo-epithelial contractile response identical to the route of intranasal administration. A breast fluid collector, preferably a breast pump, is then applied to the nipple and used to receive the expressed breast fluid.

In a preferred method involving the use of a milking pump, a negative pressure is generated on the breast to facilitate the release of oxytocin-stimulated breast fluid. Also, the breast fluid can be released and collected without using a milking pump. This may require increasing the dose of oxytocin or increasing the post-administration time before the mammary fluid is completely released from the nipple. During or after the release process of the breast fluid, a biological sample is collected from the released breast fluid, the sample comprising whole breast fluid, whole cells of breast fluid, cell fragments, cell membranes, selected fluids, cells or other cells. It can consist of solid fractions as well as proteins, glycoproteins, peptides, nucleotides (including DNA and RNA polynucleotides) and other similar biochemical and molecular components of breast fluid.

In a related aspect of the present invention, there is provided a method of determining the presence or amount of a marker of breast disease, particularly a breast cancer marker, in a biological sample obtained from the breast organ of a mammalian patient. These methods include administering oxytocin or an oxytocin analog to the patient intranasally, intramuscularly or intravascularly in an amount sufficient to stimulate the release of breast fluid in the mammalian patient. Once oxytocin has reached the target alveolar tract tissue and sufficient post-administration time has elapsed to stimulate said tissue, breast fluid may be collected directly from the nipple, or the breast may be pumped as described above. And collecting a biological sample from the released breast fluid. After the sample is collected, a bioassay is performed to determine the presence and / or amount of breast disease markers in the sample.

Suitable bioassays in this regard include assays that detect known markers of breast infection, e.g., using immunological or other suitable probes, to include specific antigens and bacterial and viral pathogens. Include assays that detect other markers expressed by the selected pathogen. A more preferred bioassay would detect individual markers or a panel of markers for benign breast tumor, precancerous breast disease, and / or breast cancer.Such assays include immunological or other suitable probes. It is used to detect specific antigens and other markers expressed by benign, precancerous, and / or cancerous alveolar duct cells of the breast.

In yet another aspect of the present invention, there is provided a kit for determining the presence or amount of a marker of breast disease, particularly a breast cancer marker, in a biological sample obtained from the breast organ of a mammalian patient. The kit includes a formulation of oxytocin in a biologically suitable carrier. Preferably, the oxytocin formulation is an oxytocin solution prepared in a nasal spray applicator. The kit also includes a collection device for collecting a biological sample from the expressed breast fluid, the collection device extending from a reservoir of the sample fluid to a solid phase medium that can be directly incorporated into a solid phase bioassay. Can be range.

In this regard, there is provided a reservoir or solid phase collection device that applies a negative pressure to the udder to promote the development of breast fluid from the nipple after stimulation of oxytocin and is incorporated into a milking pump for collection of biological samples. An optional milking pump can be provided that achieves the dual purpose of: In a particularly preferred embodiment of the invention, the kit comprises a composition and / or a device for detecting the presence or amount of a marker for breast disease in a biological sample, such as an immunology that binds to or reacts with a breast cancer marker. Includes molecular or molecular probes.

As described above, the present invention provides a method for obtaining a biological sample from breast fluid. Preferably, these methods are non-invasive, meaning that they are non-surgical and do not involve penetration of the breast by a needle or other intrusion device. In order to achieve a non-invasive sample collection method, in particular, the present invention is useful for stimulating the release of breast fluid from a patient's nipple when applying a milking pump to promote the development of breast fluid. It relies on the administration of large amounts of the peptide hormone oxytocin to mammalian patients. Preferably, the oxytocin preparation is administered intranasally and is administered in an intranasally effective amount to stimulate the release of mammary fluid from the nipple.

Oxytocin is a pituitary-derived peptide hormone that is spontaneously released into the bloodstream of lactating women in response to aspiration and stimulates contraction of myoepithelial cells in mammalian alveoli and ducts And cause milk injection. Cobo, J. Perinat. Med., 21: 77-85, 1993. This drug has also been widely used to stimulate labor in pregnant women due to its activity to stimulate uterine contractions. Satin et al., Am. J. Obstet. Gynercol., 166: 1260-1261, 1992. For these reasons, the pharmacology of oxytocin has been well studied and involves a detailed study of effective dosage, half-life and potential side effects.

Oxytocin preparations containing oxytocin in a biologically suitable liquid carrier for use in the present invention are provided for intranasal, intramuscular, or intravascular administration. The most economical oxytocin preparations, various suppliers, for example, sand (Sandoz) (Basel, Switzerland) and United States-Famakopeia (United State Pharmacopeia) available from synthetic oxytocin analogs (e.g., Pitocin ^R or Use Syntocinon ^R ). Naturally-occurring oxytocin from mammalian sources, as well as other known naturally-occurring oxytocin-like peptide analogs and their synthetic equivalents having similar activity in stimulating the contraction of alveolar myoepithelium, are of course suitable. It is. (See, for example, Amico et al., J. Clin. Endocrinol. Metab., 60: 5, 1985, which is incorporated herein by reference in its entirety).

好 ま し い For use in the methods and kits of the present invention, a preferred oxytocin preparation contains approximately 40 units of oxytocin / ml liquid carrier. Preferred liquid carriers are biologically compatible solutions, such as Ringer's lactated solution or other physiologically balanced, sterile, non-toxic and non-irritating solutions. To administer oxytocin intranasally, a standard nasal squeeze bottle is used that, when squeezed, delivers approximately 0.5 ml of the oxytocin preparation into the patient's nose.

Oxytocin is absorbed by the nasal mucosa into the systemic circulation, where it reaches and acts specifically on the myoepithelial cells surrounding the alveoli of the breast and making up the ductal wall, The fibers are contracted, forcing the existing fluid into a large tube or sinus, where the fluid is released spontaneously from the nipple onto the sample collector or is developed by further action of the milking pump. Can be The half-life of oxytocin in the human bloodstream is estimated to be extremely short, about 10-15 minutes or shorter, due to rapid removal from plasma by the kidneys, liver, and mammary gland, and to pharmacokinetics and clinical The time to target steady state is readily determined depending on the mode of administration (eg, bolus, multiple dose, or constant infusion). (Eg, Gonser, Arch. Gynecol. Obstet., 256: 63-66, 1955; and Orhue, Obstet. Gynecol., 83: 229-233, 1994, each of which is incorporated herein by reference in its entirety. Division, see).

Thus, an effective (nasally effective, intravenously effective, or intramuscularly effective) amount to cause the release of breast fluid with or without the aid of a milking pump Determining the appropriate concentration and dosage of oxytocin preparation to administer oxytocin is a matter of routine. (See, for example, the following references: Newton, Ann.NYAcad.Sci., 652: 481-483; Mena, Neuroendocrinology, 61: 722-730, 1995; Gonser, Arch.Gynecol.Obstet., 256: 63-66, 1995; Orhue, Obstet Gynecol., 83: 229-233, 1994; Satin et al., Am.J.Obstet.Gynercol., 166: 1260-1261, 1992; and Satin et al., Obstet Gynecol. , 83: 234-238, 1994, each of which is hereby incorporated by reference in its entirety).

Although not all female patients are expected to respond to nasal oxytocin stimulation, an intranasally effective amount of oxytocin for the purposes of the present invention can be readily determined. As used herein, an intranasally effective amount of oxytocin is obtained by applying oxytocin spray to at least 50% of non-lactating women with the help of a negative pressure on the nipple of 50-200 mmHg applied by a milking pump. By 45 minutes after the first dose, an amount sufficient to stimulate the release of at least 3 μl of mammary fluid intranasally. Administering a low, preliminary dose of oxytocin to the patient, e.g., administering a single spray of 40 units / ml oxytocin solution in each nostril or multiple sprays of a lower concentration oxytocin solution It may be necessary to wait and then measure the sensitivity of a particular patient, which is in fact preferred.

If there is no reaction in the initial application of the milking pump after a short period after administration for 2-15 minutes, preferably 2-5 minutes, a booster dose of oxytocin spray can be administered and the pump can be applied again . In this way, the clinician can adjust the dosage for each patient's changing sensitivity, thereby minimizing potentially adverse side effects. Also, in patients who have failed intranasal administration or are otherwise contraindicated as the preferred mode of administration, intramuscular or intravascular administration of oxytocin may be used according to the same dosage and administration principles. it can.

Once an effective dose of oxytocin is administered intranasally and the clinician allows oxytocin to reach the target alveolar tract tissue and stimulate the target alveolar tract tissue after an appropriate post-dose period, the milking pump is turned off. Applied according to well-known techniques, a negative pressure is created on the breast to facilitate the release of breast fluid. Within the scope of the method according to the invention, negative pressures of 50 to 200 mmHg are preferred, and depending on the sensitivity of the individual patient, the dose of oxytocin and other factors, these negative pressures are preferably intermittent, Hold for approximately 1-15 minutes. Also, without the aid of a milking pump, breast fluid expression can be achieved using a sample collector that receives the expressed breast fluid, as described herein.

The volume of breast fluid expressed will vary depending on various factors, such as the patient's sensitivity to oxytocin, the delivered oxytocin dose, the time and pressure of the milking pump, and other factors. . For the least sensitive breast marker assay of the present invention, a volume of 300-500 μl of released breast fluid is preferred to provide sufficient material to perform the assay, and these volumes are determined according to the methods described above. It could be obtained from a substantial proportion of treated women.

To release 300-500 μl of mammary fluid, some women will require repeated stimulation and will likely require a pool of mammary fluid samples obtained during multiple patient visits . However, for more sensitive assays of the invention, such as solid-phase immunoassays, in particular as compared to the surface or intracellular antigens of breast epithelial cells, which are naturally secreted, for example, naturally into the mammary fluid and thus do not. In the case of breast cancer markers that are expected to be present at very high concentrations, a very small sample of 3 μl or less would be appropriate to perform this assay.

の 間 Collect a biological sample from the released breast fluid during or after the process of releasing the breast fluid. Whole breast fluid, selected liquid or solid fractions of breast fluid, whole cells or components of cells of breast fluid, proteins, glycoproteins, peptides, nucleotides (including polynucleotides of DNA and RNA) and the like A range of suitable biological samples, including various biochemical and molecular components, are contemplated and will be useful within the methods of the present invention. Sample collection can be accomplished simply by placing the released breast fluid into a suitable reservoir, such as a conventional sample storage or assay container.

Also, the released breast fluid may be exposed to conventional buffers, diluents, extraction or chromatography media, filters, and the like for further processing or direct incorporation into the desired assay. The sample can be collected by stabilizing or preparing the sample.

In a preferred embodiment of the invention, the released mammary fluid is exposed to a solid phase medium, such as a glass slide for a microscope, a filter of nitrocellulose, an affinity column, a dot-blot matrix or other similar medium, and the like. The phase medium selectively absorbs, binds, filters, or otherwise absorbs the desired components of the breast fluid, e.g., bulk or purified protein, to facilitate incorporation into the desired assay. If not, it will process. As those skilled in the art will understand and readily carry out, the range of possible sample collection techniques and materials useful within the scope of the present invention will be wide and will vary with each selected assay.

The basic utility of the present invention lies in a new, non-invasive method of obtaining biological samples from breast fluid, but an effective assay for detecting and / or measuring important breast disease markers in these samples. Disclose an additional method for providing In this regard, the present invention provides a wide range of assay methods that incorporate known techniques and reagents for determining the presence and / or expression level of breast disease markers, particularly breast cancer markers, in biological samples.

When incorporated within the scope of the present invention, these methods involve administering oxytocin to a mammalian patient, preferably via intranasal administration, in an amount effective to stimulate expression of breast fluid in the patient, as described above. Administration. Once sufficient time has elapsed after administration to allow oxytocin to reach the target alveolar tract tissue and stimulate the tissue, the breast is pumped and a biological sample is collected, as described above. After the sample is collected, a bioassay is performed on the sample to determine the presence and / or amount of a selected marker of breast disease, preferably a breast cancer marker or a panel of breast cancer markers, in the sample.

As used herein, the term breast disease marker refers to a breast disease-expressing breast cell that is specifically expressed (eg, as a cell surface protein or secreted protein) by diseased breast cells. Expressed at a measurably increased or decreased level by any cell or statistically significant in association with breast disease (eg, a protein expressed by an infectious agent associated with breast disease) Or is statistically significantly, measurably increased, or expressed at a reduced level by diseased breast cells as compared to normal breast cells, or is associated with breast disease. Expression by non-disease breast cells (eg, in response to the presence of diseased breast cells or substances produced therefrom) The cells means cell fragments, proteins, peptides, glycoproteins, lipids, glycolipids or other molecules or biological material.

マ ー カ ー Breast disease markers can also include specific DNA or RNA sequences that mark deleterious genetic changes or alterations in the pattern or level of expression of genes significantly associated with breast disease. Preferred markers of breast disease include breast infection, benign neoplasia, malignant neoplasia, precancerous conditions, and conditions associated with an increased risk of cancer.

As used herein, the term breast cancer marker is defined as being specifically expressed by cancer cells, or by cells of a diseased breast, or statistically by cancer cells as compared to normal cells. Significantly increased or decreased expressed at a measurable level, or expressed by non-cancer cells in connection with cancer cells (eg, in response to the presence of cancer cells or substances produced therefrom). A) a subset of markers of breast disease, ie, proteins, peptides, glycoproteins, lipids, glycolipids, proteolipids, or other molecules or biological materials that are expressed.

BREAST CANCER MARKERS can also include specific DNA or RNA sequences that mark deleterious genetic changes or alterations in the pattern or level of expression of genes significantly associated with breast disease. In addition, breast cancer markers may be cytological features of all cells present in the breast fluid, such as nuclear inclusion or cytoplasmic structures, or uniquely expressed by or associated with cancer cells. Attributes of the staining can be included.

Among the breast cancer markers that are effective within the method of the invention, a subset is described in the following representative review articles; Porter-Jordan et al., Hematol.Oncol.Clin.North Amer., 8: 73-100, 1994; and Greiner, Pharmaceutical Tech., May, 1993, pp. 28-44, each of which is hereby incorporated by reference in its entirety. Other suitable markers are also widely known and can be readily incorporated into the methods of the invention using information and methods commonly known or available in the literature.

Preferred breast cancer markers for use within the scope of the present invention are more characterized markers that have been shown to have significant value in determining prognostic and / or treatment related variability in female patients Is included. As noted above, prognostic variability is a variability that serves to predict disease outcome, such as the likelihood and timing of recurrence or survival. Variations related to treatment predict the likelihood of success or failure of a given treatment program. Determining the presence or level of expression or activity of one or more of these markers can facilitate the differential diagnosis of patients with malignant or benign abnormalities, and provide a risk of relapse or a choice of treatment option Useful for predicting the likelihood of response.

It is important to note, however, that the present invention does not rely solely on breast disease markers that meet the stringent requirements of sensitivity and specificity that make it immediately acceptable for clinical application in human patients. Conversely, a number of breast disease markers contemplated within the scope of the present invention do not meet these stringent criteria, but nevertheless are substantially beneficial when detecting, differentially diagnosing or managing breast cancer. Provide some useful information.

Such non-clinically accepted markers are useful for immediate application within the methods of the present invention, as basic research tools and as ancillary tools in clinical applications. Beyond these immediate applications, a number of such markers are particularly useful in assay methods that analyze marker combinations and generate complementary data of greater predictive value than the data generated by individual markers alone. It is expected that it will be further developed and refined to the point of direct clinical applicability according to the methods of the present invention.

The preferred assays of the present invention are particularly focused on breast cancer markers associated with tumor development, tumor growth, angiogenesis and cancer invasion, and in this context are important for cancer risk, presence, condition or future behavior in patients. Provide important information. As described above, tumor development and tumor growth can be assessed using various cell proliferation markers, such as Ki67, cyclin D1 and PCNA. Tumor growth can also be attributed to various growth factor and hormone markers (eg, estrogen, EGF, erB-2, and TGFα), autocrine or exocrine growth factor and hormone receptors (eg, IGF and EGF receptors), or vascular It can be evaluated using a forming factor.

In addition to markers of tumor development, proliferation and growth, a number of markers provide information about the potential for cancer invasion or metastasis in cancer cells, for example, by indicating changes in cell adhesion or motility factor expression or activity. provide. Typical markers for this include cathepsin D, plasminogen activator and collagenase. In addition, the expression levels of several putative tumor "suppressor" genes, such as nm23, p53 and rb, provide important data regarding the metastatic potential or growth regulation of cancer cells. Large numbers and variables of appropriate breast cancer markers in each of these classes have been identified, and many of these have been shown to have significant value in determining prognostic and / or treatment-related variability for breast cancer .

前 Prior to or at the same time as each assay of the present invention, it is preferable to perform a preliminary evaluation to confirm the origin and / or quality of the sample. Such a preliminary assessment proves that the sample collected from the expressed breast fluid is in fact of breast origin and is not contaminated with other potential contaminants, such as sweat from the skin surrounding the nipple Focus on doing. For the purpose of demonstration of these samples, markers that are known to be present in mammalian breast fluid and are preferably highly specific for breast fluid (ie, typically A variety of assays are available to identify markers of breast fluid that are always present in the fluid and that do not contain all or most of the other potentially contaminating bodily fluids and body tissues.

However, acceptable levels of specificity for breast fluid markers within the scope of the method of the present invention are provided by markers that are simply known to be present in breast fluid, but they are not compatible with other bodily fluids. May exist in. One such marker is the enzyme lysozyme, a normal component of human serum, urine, saliva, tears, nasal secretions, vaginal sections, semen, and breast fluid.

Lysozyme (muramidase) is an enzyme that hydrolyzes beta-1,4-glycosidic bonds in the cell walls of mucopolysaccharides of various microorganisms that cause cell lysis. Quantitative determination of the lysozyme is achieved by the diffusion method well-known agar plates, the diffusion method, Carre stud, Sanofi Diagnostics (Kllestad, Sanofi Diagnostics) available from (Minnesota Chasuta) Qunatiplate ^R The instructions provided with the lysozyme test kit are described in detail, which is incorporated herein by reference in its entirety.

Other breast fluid markers for the examination of a sample that are more specific than lysozyme are preferred within the scope of the method of the invention and are within the scope of the invention based on published and generally known information. Can be easily incorporated into Most preferred among these markers are proteins and other biologicals that are specifically expressed or concentrated in breast fluid. In this regard, suitable markers of various sequences have been characterized and have already been used to develop specific antibodies, eg, affinity purified antibodies and monoclonal antibodies.

Subsequently, using these antibodies as immunological probes to determine the presence or absence and / or quantification of selected breast fluid markers to examine the origin and quality of breast fluid samples Can be. Markers of breast fluid that are of particular interest for use within the scope of the present invention include cytokines characteristically expressed by normal and cancerous breast epithelial cells, to which specific panels of antibody probes are directed. Has already been developed. (See, e.g., Nagle, J. Histochem. Cytochem., 34: 869-881, 1986, incorporated herein by reference in its entirety).

In addition, human breast epithelial antigens (HME-Ags), which are equivalent to the glycoprotein component of human lipoglobulin (HMFG) protein, are useful as markers for breast fluid, and antigens specific to these (for example, HMFG1, Unipass, UK) ) Is also available. (See the following references: Rosner et al., Cancer Invest., 13: 573-582, 1995; Ceriani et al., Proc. Natl. Acad. Sci. USA, 74: 582-586, 1982; Ceriani et al., Breast Cancer Res. Treat., 15: 161-174, 1990, each of which is incorporated herein by reference in its entirety).

生物学 To perform an assay for a marker of breast disease provided within the scope of the invention, a biological sample collected from breast fluid is generally exposed to a probe. The probe specifically binds to a selected breast disease or breast cancer marker or otherwise interacts with the marker in a detectable manner to detect the presence or absence of a breast disease or breast cancer marker in the sample. Or the amount. The probe selected for this purpose will generally depend on the characteristics of the marker for breast disease, ie, whether the marker is a polynucleotide or other substance of the protein.

好 ま し い In a preferred embodiment of the present invention, the breast disease marker is a protein, peptide or glycoprotein, all of which are effectively targeted in breast disease markers using specific immunological probes. These immunological probes can be labeled with a covalently attached label to provide a signal to detect the probe, or, for example, by a labeled secondary antibody that binds to the immunological probe and provides a detectable signal. It can be labeled indirectly.

General methods for producing non-human (eg, murine, rabbit, porcine, equine) antisera or monoclonal antibodies are well known and include, for example, a protein for a selected breast disease marker protein, a portion of a marker protein sequence. This can be achieved by immunizing an animal with a peptide synthesized in, a degradation product containing a portion of a marker protein sequence, or a fusion protein containing all or a portion of a marker protein bound to a heterologous protein or peptide.

Within various embodiments, cells producing the monoclonal antibodies are obtained from the immunized animal, rendered permanent, and screened, or first, to produce antibodies that bind to a selected breast cancer marker protein or peptide. , And are then made to be capable of permanent division. It is desirable to transfer the antigen-binding region (ie, F (ab ') ₂ or hypervariable region) of a non-human antigen into the framework of a human antibody by recombinant DNA technology to produce a substantially human molecule Will. Methods for producing such “humanized” molecules are generally well known and are described, for example, in US Pat. No. 4,816,397, which is hereby incorporated by reference in its entirety. Have been.

In addition, human monoclonal antibodies or portions thereof are first incorporated herein by reference to the human B cell cDNA library by Huse et al., Science, 246: 1275-1281, 1989 (in its entirety). According to the method described in (1), the DNA can be identified by screening for a DNA molecule that specifically binds to the selected marker for breast disease. The DNA molecule can then be cloned and amplified to obtain a sequence encoding an antibody (or binding domain) of the desired specificity.

Also, within the scope of the present invention, bivalent antibodies having independent antigen binding sites on each immunoglobulin molecule (eg, Thromb. Res. Suppl., X: 83, 1990, and The Second Annual IBC International Conference on Antibody Engineering, edited by A. George, Dec. 16-18, 1991; each of which is incorporated herein by reference), as well as individual antibodies with different specificities Panel is conceivable. Bivalent antibodies and panels of antibodies specifically used within the scope of the invention bind to two or more selected markers of breast disease and have a greater predictive value than the data generated by the individual markers alone Includes antibodies and panels of antibodies that generate complementary data.

Monoclonal antibodies are particularly useful within the scope of the present invention as probes for detecting, imaging, and / or quantifying the presence or activity of selected breast disease markers. In this regard, there is provided a monoclonal antibody that specifically binds to a selected marker of breast disease, incorporating one or more well-known labels, for example, a dye, a fluorescein tag or a radioactive label. By incorporating such labels, the antibodies can be used in routine assays to express one or more selected markers of breast disease in a biological sample that includes or is derived from breast fluid. , Localization and / or activity can be determined.

These assays determine the expression, localization and / or activity of selected markers of breast disease in test samples taken from patents known to be at risk for, or having breast disease, for breast disease Can be compared to results from a control study that detects and / or quantifies the same marker, obtained from a normal patient negative for breast disease. In this way, baselines and cut-off values can be determined according to routine methods to refine the assays of the invention and employ them for direct clinical applications.

検 出 Detection and / or quantification of breast disease markers in the biological sample of the present invention can be achieved by various methods. Preferred methods in this regard include, among others, well-known ELISA immunoassays, immunoprecipitation assays, and various solid-phase immunoassays, such as Western blotting, dot blots and affinity purification immunoassays. Comparable methods of using non-antibody probes to detect and / or quantify the expression and / or activity of breast disease markers are disclosed herein or elsewhere in the art and Are known.

Non-antibody probes suitable for use within the scope of the invention include, for example, probes of labeled nucleotides, which hybridize to DNA transcripts of oncogenes at standard or high stringency. And hybridizes to other DNA sequences that are associated with an increased risk of breast disease or that are associated with RNA transcripts that encode proteins for breast disease markers. Other suitable probes include labeled ligands, binding partners and cofactors for markers of breast disease (eg, ligands for growth factor receptors, or substrates for breast cancer associated proteases, eg, cathepsin D).

In certain preferred embodiments of the present invention, cDNA and oligonucleotide probes are used in Northern, Southern and dot blot assays to identify selected breast disease markers in cell samples collected from expressed breast fluid. The level of expression is identified and quantified. Measuring the expression levels of breast disease markers according to these provides important information relevant to prognosis and treatment to assess the development, growth and invasion of a wide range of breast diseases, for example, cancer in mammals, especially humans Will. For example, assays utilizing oligonucleotide probes facilitate early screening to assess hereditary genetic lesions associated with breast cancer and distinguish precancerous, early cancerous and similarly metastatic lesions in patients Will do.

In addition to the sample collection and assay methods described above, the present invention also provides kits and multi-container units comprising reagents and components for performing the sample collection and assay methods of the present invention. Briefly, these kits include the basic components for obtaining a biological sample from breast fluid, for example, a formulation of oxytocin in a biologically suitable carrier. Preferably, the oxytocin preparation is prepared in a nasal spray applicator and contains approximately 40 units of oxytocin / ml of a liquid carrier, said carrier being a simple, inexpensive buffered saline solution.

Preferred applicators can be any of a variety of pressurized aerosol or hand pump reservoir forms having a nozzle that directs a liquid spray of oxytocin into the patient's nares. The kit also preferably includes a collection device for collecting a biological sample from the expressed breast fluid, which collection device can be directly integrated into a solid-phase bioassay from a simple liquid reservoir. It can be in the range of a phase medium.

In this regard, it is also possible to provide any milking pump that can apply a negative pressure to the human breast and is designed to generate an intermittent or continuous negative pressure of about 50-200 mmHg in the area surrounding the nipple. More preferably, the milking pump applies a negative pressure that promotes the development of mammary fluid from the nipple after stimulation of oxytocin and includes a reservoir or solid phase collection device incorporated within the milking pump to collect the biological sample. Achieve the dual purpose of providing.

Kits for performing the assays of the present invention include suitable containers or other devices for collecting a biological sample from the expressed breast fluid. A range of suitable collection devices are contemplated, corresponding to the wide range of suitable biological samples that can be collected from the expressed breast fluid. For example, a simple sterile container or reservoir is provided for collecting whole breast fluid.

It also receives or distributes a selected liquid or solid fraction of breast fluid, receives or distributes cells or cellular components of breast fluid, or purified or bulk proteins, sugars from breast fluid. Various solid phase devices, such as microscopy, for receiving or distributing proteins, peptides, nucleotides (including DNA and RNA polynucleotides) or other similar biochemical and molecular components. Glass slides, membranes, filters, beads and similar media are provided.

A wide variety of such sample collection devices that can be readily employed for use within certain aspects of the invention are disclosed herein, or otherwise widely known, or It is described in the literature. These collection devices are components of a milking pump (e.g., a removable fluid positioned within the pump to directly receive or contact the expressed breast fluid as it is pumped) (E.g., a sump or filter) or may be provided separately (e.g., a non-integral, breast fluid and components of the breast fluid are exposed thereto to collect a biological sample for assay purposes). Target membrane, filter, affinity column or blot material).

In a more detailed aspect of the invention, the kit comprises reagents and / or devices that detect the presence and / or amount of a marker for breast disease in a biological sample, eg, an immunoassay that binds or reacts with a breast cancer marker. Includes biological or molecular probes. These possible reagents or devices are immunological and non-immunological probes that detect the presence or amount of a breast cancer marker in a biological sample. The kit may also contain suitable buffers, preservatives, eg, protease inhibitors, direct or sandwich labels to label the probe, and / or developing reagents to detect the signal from the label.

In one aspect, the kits of the present invention contain a monoclonal antibody useful for detecting and / or measuring a breast cancer marker in a sample. Such antibodies can be detected by labeling in advance or by binding to a secondary antibody optionally included in a kit. Antibody reagents can be provided in separate containers or in combination in a series of containers.

な お Within yet another aspect of the invention, the kit contains sequence-specific oligonucleotide primers for detecting a polynucleotide molecule encoding a breast cancer marker protein. Such primers can be provided in separate containers, or can be provided in a series of containers in combination with one or more primer pairs. Based on the general knowledge in this field and the description herein, the wide selection of other kits, including those containing specific instructions for performing the assays of the invention, is within the scope of the invention. Done in

The following examples are illustrative of the invention but should not be construed as limiting the invention. Example 1 Stimulation of Mammary Fluid Release by Intranasal Administration of Oxytocin in Combination with the Application of a Milking Pump A nasal solution of oxytocin surrounds the alveoli of the breast and is specific to the myoepithelial elements that make up the walls of the ducts Acts to contract their smooth muscle fibers, thus providing fluid into a large tube or sinus, where fluid is expressed from the nipple by the action of a milking pump. Nasal spray is rapidly absorbed by the nasal mucosa and enters the systemic circulation. Intranasal application of the spray preparation is effected by any practical and effective method of administration. Oxytocin half-life in the human circulatory system is very short, approximately 10-15 minutes, and oxytocin is then rapidly cleared from plasma by the kidney, liver, and mammary gland.

Due to the known effects of oxytocin on contracting the uterus, pregnant women are not treated by the methods described herein unless the benefit of the study outweighs the risk of preterm labor.

The nasal solution of oxytocin contains natural or synthetic oxytocin, or a functional analog thereof, whose concentration is determined by applying a milking pump to the nipple to promote the development of mammary fluid. The concentration that is intranasally effective in administering a selected volume of spray to stimulate fluid development. In this example, a preferred oxytocin preparation containing approximately 40 USP units of oxytocin per ml of lactated Ringer's solution is administered in a squeeze bottle held upright when the patient is sitting in the nose. I do. One or two sprays are administered into each nostril from a standard nasal squeeze bottle, which when squeezed, dispenses approximately 0.5 ml of the oxytocin solution / spray into the patient's nose when squeezed. Dispense by spray.

The number of sprays to be administered and the column, as well as the concentration of oxytocin in solution, are adjusted according to well-known pharmacokinetic principles (see, for example, Newton, Ann. NY Acad. Sci., 652: 481-483; Mena, Neuroendocrinology, 61: 722-730, 1995; Gonser, Arch.Gynecol.Obstet., 256: 63-66, 1995; Orhue, Obstet Gynecol., 83: 229-233, 1994; Satin et al., Am. J. Obstet. Gynercol., 166: 1260-1261, 1992; and Satin et al., Obstet Gynecol., 83: 234-238, 1994, each of which is incorporated by reference in its entirety. The amount of oxytocin administered to a patient) is increased by the aid of a milking pump to stimulate expression of at least 3 μl of mammary fluid in at least 50% of non-lactating female patients. It should correspond to an internally effective amount. For example, the clinician can vary the dosage for each patient's changing sensitivity, thereby minimizing potentially adverse side effects, the number of sprays delivered to the patient, and / or the It may be desirable to adjust the timing of the delivery.

In this example, a pre-dosage of a single spray of the oxytocin solution at 40 units / ml is delivered into each nostril of the patient, and the clinician at the administration is given a short period of 2-3 minutes after administration. Wait for a while. After this period, the breast pump is applied and the clinician determines whether the amount of oxytocin delivered was sufficient to stimulate the expression of the breast pump-assisted breast fluid. If no fluid develops at this stage, a booster dose of one or two sprays of oxytocin solution is administered to each nostril and the milking pump is reapplied 5-10 minutes after the booster dose.

An intranasally effective dose of oxytocin is administered, and a clinician is allowed to reach the target alveolar tract tissue over a suitable post-administration period before stimulating that tissue, then the well-known Apply a milking pump according to the method used. A negative pressure of 50-200 mmHg is applied to the teat area and is maintained intermittently or continuously, for approximately 1-15 minutes, depending on the sensitivity of the individual patient, the dose of oxytocin and other factors. Oxytocin can be administered by intramuscular or intravascular routes by well-known means (Oxytocin Injection (synthetic), USP; Wyeth-Ayerst Laboratories) to obtain the same response as intranasal administration.

に よ り By the method described above, a primary sample of breast fluid containing at least 3 μl of fluid is expressed by 50% or more of non-lactating female patients. During or after the step of developing breast fluid, a biological sample is collected from the expressed breast fluid. In this embodiment, a nitrocellulose filter is placed in a milking pump in-line with the passage of the expressed breast fluid into the pump so that the expressed fluid is in contact with the filter.

When the primary sample of expressed breast fluid contacts the filter, the cells, proteins and other desired components of the breast fluid adhere to the filter, forming a filter-bound biological sample for subsequent analysis. Other suitable biological samples include whole breast fluid samples, whole cell cytological samples, membranes or other cellular components, and proteins, glycoproteins, peptides, nucleotides and other components of primary breast fluid samples. And can be collected according to well-known principles and methods, by appropriate modifications of the above-described procedures.

Example 2 Inspection of Sample Origin and Quality Using Lysozyme Analysis It is determined that the primary or collected sample of the mammary fluid obtained by the method described above is of breast origin and is not contaminated with similar contaminants. One or more components of normal breast fluid are assayed to confirm. In this example, the enzyme normally present in breast fluid, lysozyme, is assayed in the primary breast fluid sample to facilitate confirmation that the sample is of breast origin.

Lysozyme (muramidase) is an enzyme that hydrolyzes the beta 1,4-glycoside mammary fluid in the mucopolysaccharide cell walls of various microorganisms, and its activity is easily detected using routine, inexpensive assays And can be quantified. Lysozyme is measured in primary breast fluid samples with the Contiplate Lysozyme Test kit (Kallestad, Chasta, MN).

This assay uses reagents and procedures provided by the manufacturer and described in detail in the manufacturer's instructions, except that a breast fluid sample is used instead of serum, urine or tears. Analysis of these results establishes that the sample contains lysozyme, a common component of human serum, urine, saliva, tears, nasal secretions, vaginal secretions, semen, and breast fluid.

Use a more specific assay in place of the lysozyme assay, or to supplement the results of the lysozyme assay, especially if clinical data has been collected for human patients. Other breast fluid markers for the validation of samples that are more specific than lysozyme can be readily incorporated within the scope of the present invention, based on published and generally known information. In one example, the presence of cathepsin D is performed by monoclonal antibodies and methods described in the following literature: Vetvicka et al., Biochem. Mol. Biol. Int'l., 30: 921-928, 1993 Incorporated herein by reference in its entirety).

In another embodiment, one or more human breast epithelial antigens (HME-Ags) corresponding to the glycoproteins of human milk globulin (HMFG) protein are used in a primary breast fluid sample or assay for breast cancer markers. In biological samples to be detected using probes for specific antibodies, as described in the following references: Rosner et al., Cancer Invest., 13: 573-582, 1995; Ceriani et. USA, 74: 582-586, 1982; Ceriani et al., Breast Cancer Res.Treat., 15: 161-174, 1990, each of which is incorporated by reference in its entirety. Part of this specification.

In many cases, for example, for assays for HME-Ags, where the discovery of HME-Ags indicates the origin / quality of the sample, and also the presence and / or amount of certain breast cancer markers in the sample, Example 4 As described below, sample validation assays can be incorporated into breast cancer marker assays in a single approach. In another embodiment, validation of the sample is accomplished by immunoassay of combinations (ie, multiple markers) targeting various cytokines, which are detected as a panel of cytokines expressed in a sample of breast tissue. be able to. (See Nagle, J. Histochem. Cytochem., 34: 869-881, 1986, which is incorporated herein by reference in its entirety).

One or more of these cytokines (eg, K5, K8, K18 and K19) can be measured simultaneously or individually for breast cancer assays, and the expression level of one or more of the subject cytokines in the patient (See, eg, Focus, Harvard University News Office, March 21, 1991, pp. 2-3; and Lee, Proc. Natl. Acad. Sci., 88: 1991, each of which is incorporated herein by reference in its entirety).

Example 3 Cytology in Biological Samples from Breast Fluid In this example, conventional cytological techniques are used to identify and classify breast disease from samples obtained as described in Example 1. To use. After collecting the sample in the sample collector, the central area of the clean glass slide of the microscope is contacted with the sample, the cover glass is slid over the sample, and the sample is spread along the surface of the slide. The slides are air dried and then fixed, for example, in absolute alcohol and stained with standard cytological staining reagents, such as methylene blue, hematoxylin and rosin, and other suitable staining reagents.

The slides are then examined by light microscopy for evidence of atypical proliferation of cells and cell clumps according to known methods, e.g., as described in the following literature: "Diagnosis of Non-Palpable Breast Lesions: Ultrasonographically Controlled Fine- Needle Aspiration: Diagnostic and prognostic Implications of Cytology ", by Jacqueline Mouriquand, S. Kargr Pub.Published: July 1993, ISBN: 3805557477;" Breast: Guides to Clinical Aspiration Biopsy ", Tilde S. Kline, Irwin K. Kline, Published by Igaku-Shoin MedicalPub .: May 1988, LSBN: 0896401596; "Cytopathology of the Breast (Asop Theory and Practice of Cytopathology: 5)", by Shahla Masood, American Society of Clinical Pathology: November 1998, ISBN: 0981893806; "Fine" Needle Aspiration Cytology and Its Clinical Applications: Breast and Lung ″, Philip S. Feldman, American Society of Clinical Pathology: November 1984, ISBN: 0981891846, each of which is drawn in its entirety. It is incorporated herein by.

Example 4 Immunoassay for Mammary Epithelial Antigen in Biological Samples from Mammary Fluid Human mammary epithelial antigen (HME-Ags) is a glycoprotein component of human mammary gland globulin (HMFG) and the membrane of mammary epithelial cells. Yes, they are released by breast tumors, but not by normal breast tissue. (Ceriani et al., Proc. Natl. Acad. Sci., 74: 582-586, 1977, which is incorporated herein by reference in its entirety). In this example, as described in Ceriani et al., Proc. Natl. Acad. Sci., 79: 5420-5425, 1982, which is incorporated herein by reference in its entirety. Detect some HME-Ags with molecular weights of 150, 70, and 45 kilodaltons using specific anti-HMFG or anti-human mammalian epithelial (α-HME) probes made and used in And measure.

To begin this assay, a biological sample from breast fluid collected on a filter of nitrocellulose is electrophoretically eluted into phosphate buffered saline according to standard methods to provide a test sample. Get. Also, whole breast fluid or other types of biological samples obtained from breast fluid are made up in a suitable medium or mixture to provide a test sample for the assay. Once the test sample is thus obtained, it is assayed according to the HME-Ags radioimmunoassay (RIA) method described in the following literature: Ceriani et al., Breast Cancer Res. Treat., 15: 161-174, 1990 (incorporated herein by reference in its entirety).

Briefly, the RIA involves two preliminary treatments of a biological sample to separate interfering factors: a centrifugation step to separate the fat present, and a potential immunization using polyethylene glycol (PEG). A second settling step to settle the complex. The next step involves a unique assay in which the HMFG antigen bound to a solid support (microtiter plate) is presented to a stoichiometric or lower amount of α-HME antibody, and as described above. Complete the binding of α-HME with a biological sample from the pretreated breast fluid. In the final step, the amount of α-HME bound to the HMFG antigen on the solid phase is determined by detecting the α-HME antibody with radioiodinated and affinity purified rabbit anti-mouse immunoglobulin.

The solutions used in this assay are as follows: i) Phosphate buffered saline (PBS): 176 ml of 0.05 M KH ₂ PO ₄ , 608 ml of 0.05 M Na ₂ HPO ₄ , and H ₂ O 8 g of NaCl to a volume of 1000 ml in pH 7.4, ii) RIA buffer: 0.3% Triton-X100 (Research Prod. International Corp., Mount Prospect, IL) + 0.05% in PBS. 0.1% BSA in sodium azide, iii) Detergent buffer: 0.3% Triton-X100 + 0.05% sodium azide in PBS, iv) Buffered polyethylene glycol (PEG): 6.6% in PBS PEG) (molecular weight 8000) (Sigma) + 0.05% sodium azide) Radioiodinated by the chloramine-T technique as reported (Ceriani et al., Proc. Natl. Acad. Sci., 79: 5420). -5425,1982) and diluted to 4 × 10 ⁶ cpm / ml of RIA buffer, ¹²⁵ I-labeled affinity purified rabbit anti-mouse immunoglobulin (R -. Mouse Ig) (Antibodies, Inc, California Deibusu). Rabbit polyclonal anti-HMFG antibody or rabbit anti-human mammary epithelial antibody (α-HME) was prepared and assayed as described (ibid.).

Expose and elute a control sample from normal human mammary fluid (nitrocellulose adsorbed and eluted to a nitrocellulose filter in the same manner as the test sample eluted) to generate a standard curve to evaluate the results of the assay. Or prepared as normal whole breast fluid or another selected type of sample obtained from normal breast fluid, and prepared in a suitable medium to prepare a suitable control assay sample) at 10,240 rpm, Centrifuge at 0 ° C for 7 minutes. Discard the top white band (if present) that formed on top of the sample.

A fresh 100 μg / ml solution of lyophilized and delipidated HMFG in detergent buffer (Ceriani et al., Proc. Natl. Acad. Sci., 74: 582-586, 1977) was prepared and 4 Sonication at 25 ° C. on a Sonifier Cell Disrupter 185 (Branson, Danbury, Conn.) At 25 ° C. using a double-step microtip at 10 second intervals for a total of 4 minutes ( Sonication for 10 seconds, followed by a silent period of 10 seconds).

HMFG solutions at concentrations of 0, 10, 33.3, 100, 333.3 and 1000 ng protein / ml were prepared in female spun serum, and three 180 μl aliquots of each HMFG level in normal female serum Is pipetted into a 400 μl polyethylene microfuge tube (West Coast Sci., Emeryville, CA). Add 150 μl of a 6.6% PEG solution to each microfuge tube and incubate the tubes at room temperature on a rotary shaker overnight.

Centrifuging 300-350 μl of the eluted nitrocellulose filtrate solution (or alternatively, breast fluid or other assay sample substitute) in a 400 μl microfuge tube at 10,240 rpm, 10 ° C. for 5-7 minutes Process the test sample in a comparable manner. The microfuge tube is then cut with a razor blade below the white band formed by the serum (if present) and 180 μl of the remaining serum is transferred into a new microfuge tube. Then 150 μl of a 6.6% PEG solution is added to each microfuge tube and the tubes are incubated overnight on a rotary shaker at room temperature.

Day 2 (1) Dilute α-HME to its appropriate concentration in detergent buffer. The antibody solution has a stoichiometric amount or less of α-HME for 6 ng of HMFG protein equivalent (protein equivalent). 6ng of HMFG is covalently attached to microtiter plates by the methylated BSA technique as previously described by Ceriani in the following references: Kennet et al. Eds., Monoclonal Antibodies and Functional Cell Lines, Plenum Press, New York , 1984, pp. 398-402, which is incorporated herein by reference in its entirety.

(2) On the second day, to process the test and control samples, the microfuge tubes are centrifuged in a SHMT rotor at 10,240 rpm, 10 ° C. for 7 minutes in a Sorvall RC5C centrifuge. In triplicate, 55 μl of the supernatant is pipetted into the wells of an empty microtiter plate (Dynatech, Alexanderia, VA) and the sedimented pellet is left undisturbed. Add 25 μl of a 6.6% PEG solution to each well. Further, the alpha-HME diluted in 30μl detergent buffer was added to each well and avoid evaporation nonporous Scotch (Scotch ^R) tape was placed over the wells. The microtiter plate is then incubated overnight at room temperature on a rotary shaker.

Day 3 (1) Centrifuge the microtiter plate at room temperature for 30 minutes (3000 rpm) to sediment suspended particulate matter.
(2) Add 50 μl of RIA buffer to the wells of a microtiter plate containing 6 ng of IIMFG and aspirate for 5 minutes.
(3) Save the entire contents of the microtiter plate from 1), reserved for PEG-induced sedimentation and already pelleted, to another set of microtiter plates containing 6 ng HMFG / well as described above. Carefully transfer wells (Days 2 and 1).

(4) Incubate the microtiter plate for 3 hours while rotating at room temperature.
(5) Wash plates 5 times with RIA buffer using Dynadrop SR-1 automatic dispenser from Dynatech.
(6) Then add radioiodinated, affinity purified rabbit anti-mouse immunoglobulin diluted in 50 μl / well RIA buffer.
(7) Cover the plate with tape and incubate for 2 hours with rotation at room temperature.
(8) Wash the plate 5 times with RIA buffer.
(9) Cut the wells and count with a gamma counter.

The results of these assays are comparable in range and value to the data provided by a serum assay performed on the HME-Ag breast cancer marker by Ceriani et al., Breast Cancer Res. Treat., 15: 161-174, 1990. Important information about the presence and / or condition of cancer in a patient. By selecting patient and control samples, generating and evaluating comparable data according to the techniques of Ceriani and co-workers, the assays of the invention can also be used in direct clinical applications. Easily employed to measure prognostic and treatment-related variability in breast cancer patients. The reagents and conditions of this assay can, of course, be substituted or adjusted depending on various expected variations by applying well-known immunological methods and principles.

Example 5 Competitive Radioimmunoassay of Impermeable Binding Proteins in Biological Samples from Breast Fluid The breast epithelial antigen contained in a biological sample from breast fluid obtained according to the method of the present invention comprises: Instead of binding to a stoichiometric amount or less of the monoclonal antibody Mc5 against a solid phase bound antigen known as a non-permeable G protein (NPGP) contained in HMFG This competitive radioimmunoassay is based.

Bind HMFG to solid support and expose to Mc5 antibody during incubation to bind antibody to NPGP antigen in solid phase bound HMFG. The ability to compete for binding of Mc5 to NPGP antigen in solid phase bound HMFG ultimately tests the presence and / or level of NPGP in a biological sample. This is detected and / or measured using a radiolabeled goat anti-mouse antibody that binds to and labels the Mc5 antibody probe.

O The buffers and other solutions and reagents in this example are generally the same as those used for the HME-Ags polyclonal antibody radioimmunoassay described in Example 4 above. To prepare the test sample for this assay, a biological sample from the mammary fluid contained on a nitrocellulose filter was electrophoresed into phosphate buffered saline according to standard methods. To elute.

全 Alternatively, whole breast fluid or other types of biological samples obtained from breast fluid can be composed in a suitable medium or mixture to provide a test sample for the assay. Once the test sample is thus prepared, it is described in Cerianiet al., Breast Cancer Res. Treat., 15: 161-174, 1990, which is incorporated herein by reference in its entirety. According to the existing NPGP / Mc5 radioimmunoassay (RIA) method, it is assayed as follows:

1) 400 μl of pooled normal female mammary fluid (nitrocellulose adsorbed and exposed to a nitrocellulose filter in the same manner as the eluted test sample and eluted, or normal whole or normal breast fluid Prepare the test sample by preparing it as another selected type of sample obtained from the above and making up the test sample in a suitable medium or mixture) and prepare an appropriate control sample at a concentration of 1: 6 in RIA buffer. Dilute to 2.4 ml with liquid.

2) A solution of 500 μg / ml lyophilized HMFG was prepared in 1 × PBS containing 0.3% Triton-X100, 0.05% sodium azide, and Sonifier Cell Disrupter 185 (Branson (Danbury, Conn.) At 25 watts using a dual step microtip for 4 minutes (10 seconds sonication, then 10 seconds silent period, 4 ° C.).

3) by using 2.4 ml of 1: 6 normal female serum and increasing the amount of HMFG (0, 0.25, 2.5, 25, 50 μg / ml HMFG as described in Example 4) Prepare a solution to generate a standard curve.
4) Dilute each test assay sample 1: 6 with RIA buffer (40 μl serum / 200 μl RIA buffer) to form diluted test assay sample and vortex.

5) To contain less than stoichiometric amount of antibody for 100 ng protein / well of HMFG covalently attached to microtiter plates prepared as described by Ceriani in the following reference: To prepare a Mc5 stock solution: Kennet et al., Ed., Monoclonal Antibodies and Functional Cell Lines, Plenum Press, New York, 1984, pp. 398-402, which is hereby incorporated by reference in its entirety. Is done.

6) Add 200 μl RIA buffer to each well of 100 ng HMFG, then aspirate 5 minutes later.
7) To generate a standard curve, add 30 μl HMFG standardization solution (as in 3 above) to 100 ng probe / well HMFG microtiter wells in quadruplicate experiments.
8) Add 30 μl of diluted test assay sample (or 30 μl of breast fluid or other test assay sample alternative) to 100 ng probe / well HMFG microtiter well in triplicate.

9) Add 20 μl of Mc5 stock solution to each well.
10) Cover the microtiter plate with non-porous Scotch® tape and incubate overnight on a rotary shaker at room temperature.
11) The next day, aspirate the wells and wash 5 times with RIA buffer.
12) Disperse 50 μl of 200,000 cpm / 50 μl of ¹²⁵ I goat anti-mouse antibody in each well. The wells are covered with non-porous tape and placed on a rotary stirrer for 3 hours at room temperature.
13) Wash wells 5 times with RIA buffer.
14) Cut the wells and count the radioactivity with a gamma counter.

The results of these assays are comparable in range and value to the data provided by serum assays performed on NPGP breast cancer markers by Ceriani et al., Breast Cancer Res.Treat., 15: 161-174, 1990. Important information about the presence and / or condition of cancer in By selecting patient and control samples, generating and evaluating data according to the methods of Ceriani and co-workers, the assays of the invention can be easily adapted for use in direct clinical applications. Thus, prognostic and treatment-related variability in breast cancer patients could be measured. As those skilled in the art will appreciate, the reagents and conditions of this assay can be replaced or adjusted depending on various expected variations by applying well-known immunological methods and principles. It is.

Example 6 Solid Phase Immunoassay for Mucinous Carcinoma-Associated Antigen in Mammary Fluid In this example, a sensitive solid phase immunoassay was used to detect mucus in a biological sample from breast fluid obtained according to the method of the present invention. Detect carcinoma-associated antigen (MCA). Uses the antibody bead immunoassay kit provided by Hoffman-La Roche (Basel, Switzerland) and is provided by the manufacturer and further includes Eskelinen et al., Anticancer Res., 9: 437-440, MCA concentrations are measured using the reagents and techniques described in detail in 1989.

Briefly, expressed mammary fluid and standard test assay samples are first incubated with MCA monoclonal antibody beads, and then, after appropriate washing, an enzyme (horseradish peroxidase) labeled secondary antibody is added. During the second incubation, the anti-MCA enzyme conjugate attaches to the antibody-antigen conjugate on the beads. Excess conjugate is removed by washing, and finally the enzyme substrate is added and the color developed is recorded.

The format of the solid-phase assay provided in this example is adapted for use in other assays with a wide array to detect and / or measure other cancer markers in addition to the MCA marker with enhanced sensitivity can do. In addition, the results of these assays are evaluated together with the results of complementary assays that detect and / or measure different markers and are exemplified by the combined MCA / CA15-3 assay described in the following references: Thus, more accurate information about the presence and / or status of cancer in a patient can be generated: Eskeinen et al., Anticancer Res., 9: 437-440, 1989; see also the following references: Eskeinen et al., Anticancer Res., 8: 665-668, 1988, each of which is incorporated herein by reference in its entirety.

Example 7 Western Analysis of Proteins from the Cellular Fraction of Human Mammary Fluid Using Polyclonal and Monoclonal Antibodies to Detect Vasopressin Various assays focused on a sample of cells from human breast fluid are provided by the present invention. Is done. In general, these assays rely on the isolation of cells, membranes or other cellular components from whole breast fluid expressed according to the methods described above by standard separation methods (eg, centrifugation, sucrose gradients, and others).

Biological samples containing whole cells from expressed breast fluid are particularly useful for cytological and cytochemical tests to detect and evaluate breast cancer in patients. Biological samples containing a fraction of purified cell membranes from human breast fluid are, for example, expressed in this way by intracellular or as membrane-bound proteins by cells of the alveolar duct and thus secreted proteins. It is particularly useful for detecting and / or measuring breast cancer markers that are not easily detected in the liquid fraction of the fluid.

This example detects the peptide hormone vasopressin in a biological sample from breast fluid using a method taken from North et al., Breast Cancer Res.Treat., 34: 229-235, 1995. Focus on how to do. Specifically, this assay uses a test sample of crude protein isolated from a pooled sample of cells obtained from expressed breast fluid. Cells are separated from whole mammary fluid according to standard methods, and crude protein is extracted from cells by sonication in 100 volumes of 0.1 M HCl. The resulting protein suspension is then centrifuged at 1500 xg for 10 minutes at ambient temperature, and the soluble protein is sedimented with 40% TCA. The protein is sedimented by centrifugation at 10,000 xg for 2 minutes. The TCA is removed from the pellet by ether washing (x2).

The protein was resuspended in 0.1 M Tris HCl (pH 8.7), reduced with mercaptoethanol at 100 ° C. for 5 minutes (and in some cases S-alkylated with N-ethylmaleimide), and extracted from Laemeli, Nature, 227: 680-685, subjected to 15% SDS-PAGE electrophoresis at pH 9.3 using the method of 1970, which is incorporated herein by reference in its entirety. The separated proteins are then electrophoretically transferred to Immobilon PVDF membrane (Millipore, Bedford, Mass.) With 20 mM Tris glycine, pH 8.0.

These membranes were blocked with a 5% non-fat milk solution, washed with PBS containing 0.5% Triton (1 × 15 min, 2 × 5 min), a preparation of mouse monoclonal antibody against VP-HNP, VP Rabbit polyclonal antibody to VAG, or ubiquitous mouse or rabbit IgG (negative control) (for a description of antibodies and antibody preparations, see North et al., Breast Cancer Res. Treat., 34: 229-). 235, 1995, which is incorporated herein by reference in its entirety) at ambient temperature for 1 hour.

After a second wash in PBS-Triton (1 × 15 min, 2 × 5 min), membranes were separated with goat anti-mouse IgG-horseradish peroxidase conjugate or goat anti-rabbit IgG-horseradish peroxidase conjugate, respectively. Incubate for hours, then wash with PBS-Triton (1 × 15 minutes, 4 × 5 minutes). The X-ray film is exposed for 10 seconds to 5 minutes using the ECL western blot detection system to visualize the immunoreactive protein. The pre-stained SDS-PAGE standard protein is used as a molecular size marker.

Recent studies suggest that vasopressin is widely expressed in breast cancer. North et al., Breast Cancer Res. Treat., 34: 229-235, 1995. These and other results indicate that vasopressin and its relatives are important markers of breast cancer and can be readily detected by immunoassay of proteins isolated from breast tumor cells. Therefore, the results of this example, using cell samples isolated from human breast fluid, are also expected to generate important information regarding the presence and / or status of cancer in patients.

Example 8 Quantification of Carcinoembryonic Antigen in Biological Samples from Mammary Fluid by Dot Immunoblot Assay Useful for measuring low levels of breast cancer markers and only a small volume of expressed mammary fluid One of the more sensitive assays of the present invention, which is useful for detecting markers when available, is the dot immunoblot assay. In this example, carcinoembryonic antigen (CEA) is measured in whole breast fluid using the Elmotech anti-CEA monoclonal antibody kit (Mochid Pharmaceutical Co., Tokyo, Japan). Briefly, the anti-CEA monoclonal antibody is diluted to the appropriate concentration and coated on a plastic film.

す Rub aliquots (4 μl) of standard CEA solutions (0, 100, 200, and 500 ng / ml) or assay samples of expressed mammary fluid onto the immunization film. Prepare assay standards from the purified antigen preparation according to the manufacturer's instructions for the Elmotech kit. If necessary, 1000 ng / ml CEA is used as a standard. After drying at room temperature, the film is exposed to a peroxidase-conjugated anti-CEA antibody for 20 minutes at room temperature. The film is then thoroughly washed with 1 M saline containing 0.5% (v / v) Tween 20.

ト リ Visualize enzymatic reactions using trimethylbenzidine as a pigment former. The developer solution consisted of 0.05 mM trimethylbenzidine and 0.01% hydrogen peroxide in Mcllvain buffer (0.1% 1M phosphate-citrate buffer) pH 5.0 containing 10% methanol. The concentration of CEA in the breast fluid assay sample is determined by comparing the color components to the corresponding standards.

The assays disclosed in this example, and related assays that incorporate antibodies into other tumor markers in addition to CEA, are useful for measuring low levels of breast cancer markers and detect markers in restricted volumes. Useful to The results of these assays will generate important information that determines prognostic and treatment-related variability in breast cancer patients. As the skilled artisan will appreciate, the reagents and conditions of this assay can be substituted or adjusted according to well-known immunological methods and principles, depending on various expected variations. .

Example 9 Detection of the Activity of Procathepsin D and Cathepsin D in Biological Samples from Breast Fluid Cathepsin D is a lysosomal protein It is an aspartic protease. In this example, proteins are immunoprecipitated from whole breast fluid or from cell lysates from breast fluid using a polyclonal antibody against procathepsin D, generally according to the methods described in the following references: Chemically detect: Vetvicka et al., Biochem. Mol. Biol. Int'l., 30: 921-928, 1993 (incorporated herein by reference in its entirety). Detect cathepsin D protease activity separately or as a complementary assay, and according to the method disclosed in Vetvicka et al. (Id.).

Briefly, whole pooled mammary fluid (preferably 3 ml, if available) was mixed with 3 ml of buffer A (50 mM Tris HCl, 5 mM CaCl ₂ , 1 mM MgCl ₂ , 500 mM NaCl, pH 7. Dilute in 2). The suspension is centrifuged at 10,000 g for 30 minutes. The resulting aqueous phase is centrifuged again under the same conditions. The soluble portion (approximately 4.5 ml in total) was loaded onto a 1 ml column of Concanavalin A Sepharose (Pharmacia, Uppsala, Switzerland) equilibrated in buffer A, washed with buffer A and the retained protein was washed. Elute with 0.75M methyl α-D-mannopyranoside.

Fractions (250 μl) are analyzed for cathepsin D activity by Western blot and silver staining electrophoresis using the ¹⁴ C hemoglobin assay as described in the following literature: Lin et al., J. Biol. Chem., 264: 4482-4489, 1989, which is incorporated herein by reference in its entirety. Human milk procathepsin D is inhibited by adding 2 μl of 1 mM pepstatin (Boehringer Mannheim, Germany) dissolved in methanol to the reaction mixture.

This assay incorporates known biochemical assays in addition to or as a supplement to immunological assays to evaluate biological samples from breast fluid to determine cancer related variability. Only one example of many possible embodiments of the present invention is provided. The basic methods provided herein for obtaining samples from human breast fluid make these assays easily adaptable to a wide range of clinical applications, and within the scope of non-invasive screening protocols, the subject breast cancer markers Can be detected and / or measured.

As those skilled in the art will appreciate, other embodiments and variations of the invention using the same inventive concepts described herein are possible. More particularly, useful and extensively expanding sequences of breast cancer markers (including protein, DNA and RNA sequences and other markers) and probes (including immunological, nucleotide and biochemical probes). Are readily available for adaptation and use within the methods and kits of the present invention. Are these markers and probes cited and incorporated within the disclosure of the present invention, or published elsewhere in the literature, or described in literature well known in the art? Or has been mentioned.

Among these known, emerging markers and probes, useful examples within the scope of the present invention include Her2 (also known as erB-2 and neu). Her2 is a receptor for the growth factor of the transmembrane G protein of the family of EGF receptors encoded by a gene located on chromosome 17q, a region of frequent amplification in breast cancer cell lines. This marker is highly predictive for breast cancer and is detected in samples of cells of the invention using probes of known nucleotides to detect genetic defects in Her2 or to detect mRNA And / or can be measured to determine overexpression of Her2 associated with increased proliferation of cancer cells.

(See, for example, the following references: Visscher et al., Edited by Weisntein and Graham, Advances in Pathology and Laboratory Medicine, Vol. 5, St Louis, Mosby Yuear Book, 1992, pp. 123-161; Barbareschi et. al., Am. J. Clin. Path., 98: 408-418, 1992; Slamon et al., Science, 235: 177-182, 1987; each of which is incorporated herein by reference in its entirety. And).

Also, using available immunological probes, the level of Her2 protein is easily detected. (For an overview, see the following references: Poter-Jordan et al., Hematol. Oncol. Clin. North Amer., 18: 73-100, 1994 and the article cited therein at page 80, Each is hereby incorporated by reference in its entirety). Additional markers for use within the scope of the invention include EGF and receptors for EGF, for which immunological and non-immunological probes and assays readily adaptable within the scope of the invention include Poter. -Jordan et al., Hematol. Oncol. Clin. North Amer., 18: 73-100, pages 80-81 and the articles cited therein, each of which is incorporated herein by reference in its entirety. Which is part of the Wikipedia).

Additional examples of growth markers, growth factors and receptors, proteases, adhesins, angiogenic factors, oncogenes and tumor suppressor genes that can provide useful breast cancer markers and probes within the methods and kits of the invention are described below. Including: Ki67 growth factor, cyclin D1, proliferating cell nuclear antigen, transforming growth factor α, tissue plasminogen activator, insulin growth factor receptor, collagenase type IV, laminin receptor, integrin, p53, rb, nm23, c- myc, heat shock protein, prolactin, neuron-specific enolase, IR-14, KA1, KA14, alpha-lactalbumin, actin, and many others, the present invention of which the majority are reviewed in: Available immunological and non-immune, easily adaptable to It listed with the biological probes and assay:

Poter-Jordan et al., Hematol.Oncol.Clin.North Ame., 18: 73-100, 1994 and the articles cited therein, each of which is hereby incorporated by reference in its entirety. You. The adaptation of these markers and probes within the scope of the present invention is readily accomplished based on the teachings provided herein and in the cited references, supplemented by knowledge in the art. Therefore, the present invention is not limited by the above description, but its scope is determined by the following claims.

Claims (69)

A sample collection device for collecting a biological sample from a breast organ of a patient, comprising:
A breast adaptation member for application to a patient's breast to establish a fluid connection between the breast adaptation member and the nipple or intralobular conduit of the breast;
Pressure changing means coupled with the breast-compatible member to alter pressure at the surface of the nipple of the breast or in the intralobular conduit to induce or promote breast fluid expression from the nipple or intralobular conduit And a solid phase sample collection medium present in fluid communication with said breast compatible member for receiving a sample of expressed breast fluid from said teat or intralobular conduit. The sample collection device of claim 1, wherein the pressure changing means comprises a vacuum pump present in gas communication with the breast adaptation member for applying a negative pressure on the nipple or in the intralobular conduit. The solid sample connection medium in fluid communication with the breast compatible member selectively adsorbs and binds the desired components of breast fluid for incorporation into an assay for detecting breast patient markers, or the other. The sample collection device according to claim 1, wherein the sample is processed. The solid phase sample collection medium is in fluid contact with the nipple or intralobular conduit when the breast compatible member is applied to the breast and a positive or negative pressure is generated by the negative pressure changing means. 2. The sample collection device according to 1. The sample collection device according to claim 1, wherein the solid-phase sample collection medium is fluidly connected to a breast pump and is integrally or detachably disposed. The solid-phase sample collection medium is a microscope glass slide, a capillary, a collection tube, a column, a micro-column, a well, a plate, a membrane, a filter, a resin, an inorganic matrix, a bead, a granular chromatography medium, a plastic microparticle, and a latex particle. The sample collection device of claim 1, wherein the sample collection device is selected from the group consisting of: a coated tube, a coated mold, a coated bead, a coated matrix, or a combination thereof. The sample collection device according to claim 1, wherein the solid-phase sample collection medium includes a filter or a membrane. The sample collection device according to claim 1, wherein the solid sample collection medium comprises a nitrocellulose filter or a membrane. The sample collection device of claim 1, wherein the solid phase sample collection medium functions to receive or split cells, cell components, purified or bulk proteins, glycoproteins, peptides or nucleotides from breast fluid. The sample collection of claim 1, wherein the biological sample is selected from the group consisting of whole breast fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, nucleotide components of breast fluid, or combinations thereof. apparatus. The biological sample comprises one or more breast disease markers selected from the group consisting of proteins, peptides, glycoproteins, lipids, glycolipids, DNA polynucleotides, RNA polynucleotides, or combinations thereof. Item 4. The sample collection device according to Item 1. The biological sample is Ki67 growth factor, cyclin, proliferating cell nuclear antigen, estrogen, epidermal growth factor, transforming growth factor α, tissue plasminogen activator, insulin growth factor receptor, epidermal growth factor receptor, collagenase , Laminin receptor, integrin, p53, rb, nm23, ras, c-myc, heat shock protein, prolactin, neuron-specific enolase, IR-14, KA1, KA14, alpha-lactalbumin, actin, CEA, HME antigen, MCA 2. The sample collection device according to claim 1, comprising one or more breast disease markers selected from the group consisting of, PSA, vasopressin and cathepsin D, or a combination thereof. A non-invasive method for obtaining a biological sample from a patient's breast organ, comprising:
A breast adaptation member for application to a patient's breast to establish a fluid connection between the breast adaptation member and the nipple or intralobular conduit of the breast;
Pressure changing means coupled with the breast-compatible member to alter pressure at the surface of the nipple of the breast or in the intralobular conduit to induce or promote breast fluid expression from the nipple or intralobular conduit And said solid biological sample collection medium in fluid communication with said breast-compatible member for receiving a sample of expressed breast fluid from said teat or intralobular conduit. Applying a sample collection device for collecting a sample; and simultaneously with or subsequent to the development of said breast fluid, one or more of said expressed breast fluid, or on or within a solid phase sample collection medium. Collecting a biological sample comprising a component thereof comprising a breast disease marker. 14. The method of claim 13, wherein the pressure changing means comprises a vacuum pump present in gaseous communication with the breast-compatible member or intralobular conduit applying a negative pressure on the exterior of the nipple or in the intralobular conduit. Method. The solid phase sample collection medium is in fluid contact with the nipple or intralobular conduit when the breast compatible member is applied to the breast and a positive or negative pressure is generated by the negative pressure changing means. 13. The method according to 13. 14. The method of claim 13, wherein the solid sample collection medium is integrally or removably disposed in fluid communication with a breast pump. The solid-phase sample collection medium is a microscope glass slide, a capillary, a collection tube, a column, a micro-column, a well, a plate, a membrane, a filter, a resin, an inorganic matrix, a bead, a granular chromatography medium, a plastic microparticle, and a latex particle. 14. The method of claim 13, wherein the method is selected from the group consisting of: a coated tube, a coated mold, a coated bead, a coated matrix, or a combination thereof. 14. The method of claim 13, wherein said solid sample collection medium comprises a filter or a membrane. 14. The method of claim 13, wherein said solid sample collection medium comprises a nitrocellulose filter or membrane. 14. The method of claim 13, wherein the solid phase sample collection medium functions to receive or split cells, cell components, purified or bulk proteins, glycoproteins, peptides or nucleotides from breast fluid. 14. The method of claim 13, wherein said biological sample is selected from the group consisting of whole breast fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, nucleotide components of breast fluid, or combinations thereof. The biological sample comprises one or more breast disease markers selected from the group consisting of proteins, peptides, glycoproteins, lipids, glycolipids, DNA polynucleotides, RNA polynucleotides, or combinations thereof. Item 14. The method according to Item 13. The biological sample is Ki67 growth factor, cyclin, proliferating cell nuclear antigen, estrogen, epidermal growth factor, transforming growth factor α, tissue plasminogen activator, insulin growth factor receptor, epidermal growth factor receptor, collagenase , Laminin receptor, integrin, p53, rb, nm23, ras, c-myc, heat shock protein, prolactin, neuron-specific enolase, IR-14, KA1, KA14, alpha-lactalbumin, actin, CEA, HME antigen, MCA 14. The method of claim 13, comprising one or more breast disease markers selected from the group consisting of, PSA, vasopressin and cathepsin D, or a combination thereof. The step of collecting the biological sample comprises contacting the expressed breast fluid with a solid sample collection medium carrying at least one or more breast disease markers from the expressed breast fluid. 14. The method of claim 13, wherein the method comprises: An assay method for determining the presence or amount of one or more breast disease markers in a biological sample obtained from a patient's breast organ, comprising:
A breast adaptation member for application to a patient's breast to establish a fluid connection between the breast adaptation member and the nipple or intralobular conduit of the breast;
Pressure changing means coupled with the breast-compatible member to alter pressure at the surface of the nipple of the breast or in the intralobular conduit to induce or promote breast fluid expression from the nipple or intralobular conduit And said solid biological sample collection medium in fluid communication with said breast-compatible member for receiving a sample of expressed breast fluid from said teat or intralobular conduit. Applying a sample collection device for collecting a sample; and simultaneously with or subsequent to the development of said breast fluid, one or more of said expressed breast fluid, or on or within a solid phase sample collection medium. Collecting a biological sample comprising a component thereof comprising a breast disease marker; and detecting the presence or amount of said one or more breast disease markers in said sample. Assay how. 26. The pressure pump according to claim 25, wherein the pressure changing means comprises a vacuum pump present in gaseous communication with the breast matching member or intralobular conduit applying a negative pressure on the exterior of the nipple or in the intralobular conduit. Assay method. The solid phase sample collection medium is in fluid contact with the nipple or intralobular conduit when the breast compatible member is applied to the breast and a positive or negative pressure is generated by the negative pressure changing means. 25. The assay method according to 25. 26. The assay method according to claim 25, wherein the solid-phase sample collection medium is fluidly connected to a breast pump and is integrally or detachably disposed. The solid-phase sample collection medium is a microscope glass slide, a capillary, a collection tube, a column, a micro-column, a well, a plate, a membrane, a filter, a resin, an inorganic matrix, a bead, a granular chromatography medium, a plastic microparticle, and a latex particle. 26. The assay method of claim 25, wherein the method is selected from the group consisting of: a coated tube, a coated template, a coated bead, a coated matrix, or a combination thereof. 26. The assay method according to claim 25, wherein the solid sample collection medium comprises a filter or a membrane. 26. The assay method according to claim 25, wherein the solid sample collection medium comprises a nitrocellulose filter or a membrane. 26. The assay method according to claim 25, wherein the solid phase sample collection medium functions to receive or split cells, cell components, purified or bulk proteins, glycoproteins, peptides or nucleotides from breast fluid. 26. The assay method according to claim 25, wherein said biological sample is selected from the group consisting of whole breast fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, nucleotide components of breast fluid, or combinations thereof. . The biological sample comprises one or more breast disease markers selected from the group consisting of proteins, peptides, glycoproteins, lipids, glycolipids, DNA polynucleotides, RNA polynucleotides, or combinations thereof. Item 26. The assay method according to Item 25. The biological sample is Ki67 growth factor, cyclin, proliferating cell nuclear antigen, estrogen, epidermal growth factor, transforming growth factor α, tissue plasminogen activator, insulin growth factor receptor, epidermal growth factor receptor, collagenase , Laminin receptor, integrin, p53, rb, nm23, ras, c-myc, heat shock protein, prolactin, neuron-specific enolase, IR-14, KA1, KA14, alpha-lactalbumin, actin, CEA, HME antigen, MCA 26. The assay method according to claim 25, comprising one or more breast disease markers selected from the group consisting of, PSA, vasopressin and cathepsin D, or a combination thereof. The step of collecting the biological sample comprises contacting the expressed breast fluid with a solid sample collection medium carrying at least one or more breast disease markers from the expressed breast fluid. 26. The assay method according to claim 25, wherein A non-invasive method for obtaining a biological sample from a patient's breast organ, comprising:
Applying a positive or negative pressure to the patient's breast, nipple or intralobular conduit to induce or promote mammary fluid expression from the nipple or intralobular conduit;
Applying the breast matching member to the breast of the patient to establish a fluid connection between the nipple or intralobular conduit and the breast matching member;
Providing a solid-phase sample collection medium present in fluid communication with the breast-compatible member to receive a sample of expressed breast fluid from the nipple or intralobular conduit; and simultaneously with or to the expression of the breast fluid Subsequently collecting a biological sample comprising said expressed breast fluid or a component thereof comprising one or more breast disease markers on or within a solid phase sample collection medium. . Applying a positive or negative pressure to the breast, nipple or intralobular conduit, wherein the vacuum pump is in gas communication with the breast compatible member to apply a negative pressure to the nipple and / or intralobular conduit. 38. The method of claim 37, comprising activating. The solid phase sample collection medium is in fluid contact with the nipple or intralobular conduit when the breast compatible member is applied to the breast and a positive or negative pressure is generated by the negative pressure changing means. 37. The method of claim 37. 38. The method of claim 37, wherein the solid sample collection medium is integrally or removably disposed in fluid communication with a breast pump. The solid-phase sample collection medium is a microscope glass slide, a capillary, a collection tube, a column, a micro-column, a well, a plate, a membrane, a filter, a resin, an inorganic matrix, a bead, a granular chromatography medium, a plastic microparticle, and a latex particle. 38. The method of claim 37, wherein the method is selected from the group consisting of: a coated tube, a coated mold, a coated bead, a coated matrix, or a combination thereof. 38. The method of claim 37, wherein said solid sample collection medium comprises a filter or a membrane. 38. The method of claim 37, wherein said solid sample collection medium comprises a nitrocellulose filter or membrane. 38. The method of claim 37, wherein the biological sample is selected from the group consisting of whole breast fluid, whole cells, cell fragments, cell membranes, proteins, glycoproteins, peptides, nucleotide components of breast fluid, or combinations thereof. The biological sample comprises one or more breast disease markers selected from the group consisting of proteins, peptides, glycoproteins, lipids, glycolipids, DNA polynucleotides, RNA polynucleotides, or combinations thereof. Item 36. The method according to Item 37. The biological sample is Ki67 growth factor, cyclin, proliferating cell nuclear antigen, estrogen, epidermal growth factor, transforming growth factor α, tissue plasminogen activator, insulin growth factor receptor, epidermal growth factor receptor, collagenase , Laminin receptor, integrin, p53, rb, nm23, ras, c-myc, heat shock protein, prolactin, neuron-specific enolase, IR-14, KA1, KA14, alpha-lactalbumin, actin, CEA, HME antigen, MCA 38. The method of claim 37, comprising one or more breast disease markers selected from the group consisting of, PSA, vasopressin and cathepsin D, or a combination thereof. The step of collecting the biological sample comprises contacting the expressed breast fluid with a solid sample collection medium carrying at least one or more breast disease markers from the expressed breast fluid. 32. The method of claim 31, wherein the method comprises: Collected by artificial induction of breast fluid expression to detect the presence or absence of breast disease and / or determine prognostic and / or treatment-related variables associated with said breast disease in the patient from whom the sample is obtained A composition for determining the presence or amount of one or more breast disease markers in a biological sample of breast milk that has been subjected to, comprising:
One or more components in breast fluid collected by artificial induction of breast fluid expression, wherein said one or more components comprises whole breast fluid, whole cells, cell fragments, cell membranes, purified protein, bulk protein, The breast fluid is selected from the group consisting of peptides, glycoproteins, lipids, glycolipids, proteins, DNA polynucleotides and RNA polynucleotides, wherein the one or more components in the breast fluid include a detectable amount of one or more breast disease markers. And the presence or amount of said breast disease marker in said biological sample correlates with the presence, absence, severity or risk of said breast disease in said patient; and of said one or more breast disease markers A composition comprising a detection reagent for determining the presence or amount. 50. The composition of claim 48, wherein said biological sample of breast fluid is collected following administration of oxytocin or an oxytocin analog in an amount sufficient to induce expression of said breast fluid from said patient. 50. The composition of claim 48, wherein said biological sample of breast fluid is collected using a breast fluid collection device. The one or more breast disease markers are Ki67 growth factor, cyclin, proliferating cell nuclear antigen, estrogen, epidermal growth factor, transforming growth factor α, tissue plasminogen activator, insulin growth factor receptor, epidermal growth factor Receptor, collagenase, laminin receptor, integrin, p53, rb, nm23, ras, c-myc, heat shock protein, prolactin, neuron-specific enolase, IR-14, KA1, KA14, alpha-lactalbumin, actin, HME antigen, 50. The composition of claim 48, wherein the composition is selected from the group consisting of MCA, PSA, vasopressin and cathepsin D, or a combination thereof. 49. The composition of claim 48, wherein said biological sample of breast fluid comprises an initial volume of about 300-500 [mu] l or less of the collected material. 53. The composition of claim 52, wherein said biological sample of breast fluid comprises an initial volume of about 3 [mu] l or less of the collected material. The detection means includes an immunological probe that specifically binds to a breast disease marker, wherein the immunological probe or breast disease marker, or a combination or binding complex thereof is present in the presence or absence of the breast disease marker. 49. The composition of claim 48, wherein the composition provides a detectable signal for detecting or measuring the amount. 56. The composition according to claim 54, wherein said immunological probe comprises a monoclonal antibody. 56. The composition of claim 54, wherein said detection means comprises a number of immunological probes that bind to the same or different breast disease markers. 56. The composition of claim 54, wherein said detection means can be incorporated into an ELISA immunoassay, an immunoprecipitation assay, or a solid phase immunoassay selected from a Western blot, a dot blot and an affinity purification immunoassay. 50. The composition according to claim 48, wherein said detection means comprises a labeled nucleotide probe that hybridizes to said breast disease marker under standard or high stringency. 59. The composition of claim 58, wherein said nucleotide probe comprises a cDNA or oligonucleotide that hybridizes under standard or high stringency to one or more breast disease markers. 請求 The composition of claim 58, wherein said detection means can be incorporated into a Northern, Southern or dot-blot assay. 59. The composition of claim 58, wherein said breast disease marker comprises a DNA or mRNA polynucleotide encoding a protein associated with said breast disease. 49. The composition of claim 48, wherein said means for detecting comprises a labeled probe selected from a ligand, a binding partner, or a cofactor for a breast disease marker. The desired components of the breast fluid are selectively adsorbed, bound, filtered, or otherwise treated for incorporation into an assay to detect or measure the one or more breast disease markers. 50. The composition of claim 48, further comprising a solid phase sample collection medium. 64. The composition of claim 63, wherein the solid phase sample collection medium is adapted for an integral or removable configuration in fluid communication with the device for collecting breast fluid. The solid-phase sample collection medium is a microscope glass slide, a capillary, a collection tube, a column, a micro-column, a well, a plate, a membrane, a filter, a resin, an inorganic matrix, a bead, a granular chromatography medium, a plastic microparticle, and a latex particle. 64. The composition of claim 63, wherein the composition is selected from the group consisting of: a coated tube, a coated mold, a coated bead, a coated matrix, or a combination thereof. 64. The composition of claim 63, wherein said solid sample collection medium comprises a filter or a membrane. 64. The composition of claim 63, wherein said solid sample collection medium comprises a nitrocellulose filter or membrane. 64. The composition of claim 63, wherein said solid phase sample collection medium functions to receive or split cells, cell components, purified or bulk proteins, glycoproteins, peptides or nucleotides from breast fluid. The solid phase sample collection medium selectively adsorbs and binds a desired component in the breast fluid for incorporation into a desired assay to detect the one or more breast disease markers. 64. The composition of claim 63, which functions to filter, filter, or otherwise treat.