JP2005516217A - Methods and reagents for rapid and effective isolation of circulating cancer cells - Google Patents

Abstract

Methods and compositions are provided for detecting circulating tumor cells and assessing the cells for changes in tumor constitution related molecules.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of US patent application Ser. No. 09 / 248,388 filed on Feb. 12, 1999 and is hereby expressly incorporated by reference. This application is the following US provisional application: 60 / 074,535 filed on February 12, 1998; 60 / 110,279 filed on November 30, 1998; November 30, 1998 No. 60 / 110,202, filed on Feb. 16, 2001; No. 60 / 268,859, filed Feb. 16, 2001; No. 60 / 269,270, each filed Feb. 20, 2001, and Claim priority from 60 / 269,271. The entire disclosure of all the provisional applications mentioned above is hereby incorporated by reference and regarded as part of this specification.

The present invention relates to the field of oncology and diagnostic tests. The present invention is useful for monitoring cancer screening, staging progression, chemotherapy treatment response, cancer recurrence and the like. More particularly, the present invention provides reagents, methods and test kits that facilitate analysis and enumeration of rare cells isolated from tumor cells or biological samples. The present invention also provides materials and methods for assessing tumor status associated with molecules such as nucleic acids, proteins, and carbohydrates, thereby assisting clinicians in planned therapeutic treatment strategies.

BACKGROUND OF THE INVENTION Approximately 600,000 new cancer patients are diagnosed each year in the United States; one in five people in the country die from cancer or complications associated with its treatment. Considerable efforts are directed at improving the treatment and diagnosis of this disease.

  Most cancer patients do not die of primary tumors. Instead, they die from multiple widespread colonies established by metastases: malignant cells that dissociate themselves from the original tumor and often travel through the body to distant sites. If a primary tumor is discovered early, it can often be removed by surgery, radiation or chemotherapy or some combination of these treatments. Unfortunately, metastatic colonies are often quite difficult to detect and remove, and it is often impossible to treat all of them successfully. Therefore, from a clinical point of view, metastasis is considered the second last event in the natural progression of cancer. Furthermore, the ability of metastasis is a characteristic that uniquely characterizes malignant tumors.

Cancer metastasis involves a complex series of consecutive events. Ie:
(1) Extension from original position to surrounding tissue;
(2) penetration into body cavities and ducts;
(3) release of tumor cells for transport through the circulatory system to distant sites;
(4) Tumor re-infiltration at the site of interest; and (5) Application to a new environment to promote tumor cell survival, angiogenesis and tumor growth.

  Based on the complexity of cancer and cancer metastasis and the frustrations in the treatment of cancer patients over many years, many of them have been developed to guide treatment and develop diagnostic tests to monitor the effects of such treatment on metastasis or recurrence Attempts have been made. Perhaps such tests can also be used for cancer screening, replacing relatively coarse tests such as mammography for breast tumors and intrarectal palpation for prostate cancer. Numerous tests have been developed for this purpose over the last 20 years. One of the first attempts is the formulation of an immunoassay for carcinoembryonic antigen (CEA). This antigen appears on fetal cells and reappears on tumor cells in certain cancers. Extensive efforts have been made to evaluate the utility of testing CEA and many other tumor antigens such as PSA, CA15.3, CA125, PSMA and CA27.29. However, the appearance of such antigens in the blood is generally not predictable and is often detected when the patient is not expecting much. However, in the past few years, one trial has proven useful for early detection of cancer. That is, prostate specific antigen (PSA) for prostate cancer. When used in follow-up medical examinations and biopsies, this PSA test has played an important role in detecting prostate cancer early when best treatment is given.

  Despite the success of the PSA test, this test remains highly desirable. For example, high levels of PSA do not always correlate with cancer and do not indicate tumor metastasis potential. This may be due in part to the fact that PSA is a component of normal prostate tissue as well as other unknown factors. In addition, it has become apparent that a large proportion of prostate cancer patients continue to have local disease, which is not life threatening. Attempts have been made to determine whether prostate cancer circulates based on the desire to obtain better harmony between patients with metastatic cancer and patients with non-metastatic cancer. In addition to high PSA levels and biopsy data, the presence of circulating tumor cells will give suggestions on how to treat patients actively.

One approach to determine the presence of circulating prostate tumor cells was to test messenger RNA expression for PSA in the blood. This is done by the difficult procedure of isolating mononuclear cells from blood samples followed by isolation of all mRNA from these cells followed by reverse transcription PCR for PSA. At the present time [Gomella LG. J of Urology. 158: 326-337 (1997)], there is good between the presence of such cells in the blood and the prediction of which patients need active treatment There is no significant correlation. It is noteworthy that PCR is difficult to perform quantitatively, ie, if not impossible in many situations, that is, to quantify the number of tumor cells per unit volume in a biological sample. Furthermore, false positives are often observed using this technique. Further disadvantages are limited sensitivity and practical limitations based on the sample size of the test. Typically, this test is performed on 10 ⁵ to 10 ⁶ cells purified so as not to interfere with red blood cells. This corresponds to a practical lower limit of sensitivity of one tumor cell per 0.1 ml of blood. Therefore, there will be approximately 10 tumor cells in 1 ml of blood before the signal is detected. As further consideration, tumor cells are often genetically unstable. Thus, cancer cells with gene rearrangements and sequence changes will be missed in the PCR assay as the necessary sequence complementarity between the PCR primer and the target sequence may be lost.

In summary, useful diagnostic tests need to be very sensitive and reliable in quantification. If a blood test could be developed that could detect the presence of a single tumor cell in 1 ml of blood, it would represent an average over 3000 to 4000 total cells circulating. In an inoculation experiment that establishes a tumor in an animal, injection of 3000-4000 cells can actually lead to the establishment of a tumor. Furthermore, if 3000-4000 circulating cells represent 0.01% of the total cells in the tumor, it will contain about 4 × 10 ⁷ total cells. Tumors containing that number of cells cannot be viewed by any existing technique. Therefore, if tumor cells are found early in the cancer, the test with the above sensitivity will detect the cancer. If it is clarified that tumor cells are in some functional relationship with tumor size, quantitative tests will be useful to assess tumor burden. To date, there is no information regarding the presence of circulating tumor cells in very early cancers. Furthermore, the medical literature regarding the presence of such cells and the possibility of such information is quite questionable. The general view is that tumor cells are well confined in the early stages, and therefore there are few, if any, circulating cells early in the disease. There is also a suspicion that the ability to detect cancer cells so early provides useful information.

Based on the above, it is clear that a method for identifying those circulating cells with potential for metastasis prior to the establishment of secondary tumors, particularly in the earliest possible cancers, is highly desirable. In order to recognize this advantage that such tests over conventional immunoassays, the lower limit of the functional sensitivity of a sensitive immunoassay can be considered to be ^10-17 moles. If one tumor cell can be captured and analyzed from 1 ml of blood, the number of moles of surface receptor would be ^10-19 moles assuming 100,000 receptors per cell. Since about 300 molecules can be detected on a single cell, such assays have a functional sensitivity on the order of ^10-22 , which is very noteworthy. In isolating such rare cells, achieving that level of sensitivity and isolating them without damaging or interfering with their properties is an irresponsible task.

  Many experimental and clinical procedures use biospecific affinity reactions to isolate rare cells from biological samples. Such reactions are commonly used for diagnostic tests or for the separation of a wide range of target substances, in particular biological matter such as cells, proteins, bacteria, viruses, nucleic acid sequences and the like.

  Various methods are available for analyzing and separating the target substance based on complex formation between the substance of interest and another substance to which the substance of interest specifically binds. Separation of the complex from unbound material is accomplished gravimetrically, for example, by standing or, alternatively, centrifugation of subdivided particles or beads coupled to the target material. If desired, such particles or beads can be magnetized to facilitate the binding / release process. Magnetic particles are well known in the art and are used for immunization or other biospecific affinity reactions. See, for example, US Pat. No. 4,554,088 and [Immunoassays for Clinical Chemistry, pp. 147-162, edited by Hunter et al., Churchill Livingston, Edinburgh (1993)]. In general, any material that facilitates magnetic or gravity separation can be used for this purpose. However, it has become clear that the magnetic separation means is the method to be selected.

  Based on size, magnetic particles can be classified as large (1.5 to about 50 microns), small (0.7-1.5 μ), or colloids (<200 nm), also called nanoparticles. The third, also known as a ferrofluid or ferrofluid-like material, has many of the properties of a classic ferrofluid and is sometimes referred to herein as a colloidal, superparamagnetic particle.

  The above types of small magnetic particles are conveniently coated with biofunctional polymers (eg, proteins) and are therefore very useful for analyzes involving biospecific affinity reactions, providing very high surface area, Gives a unique reaction rate. Magnetic particles in the range of 0.7 to 1.5 microns are described, for example, in U.S. Patent Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234. No. 4,452,773; 4,554,088; and 4,659,678. Some of these particles are disclosed to be useful solid carriers for immunological reagents.

The efficiency with which magnetic separation can be achieved and the recovery and purification of magnetically labeled cells depend on many factors. They are:
The number of cells to be separated;
The receptor or epitope density of the cell;
・ Magnetic quantity per cell;
Non-specific binding of magnetic substances (NSB);
・ Technology used;
・ The nature of the container;
-Properties of the container surface;
The viscosity of the medium; and the magnetic separation device used. If the level of non-specific binding of the system is substantially constant, its purity decreases as the target population decreases.

For example, a system of 0.8% NSB that recovers 80% of the 0.25% population in the original mixture is 25% pure. On the other hand, if the initial population is 0.01% (1 target cell in 10 ⁶ unrelated cells) and NSB is 0.001%, the purity is 8%. Therefore, the higher the purity of the target substance in the sample mixture, the more specific and effective collection of the target substance. Extremely low non-specific binding is required or advantageous to facilitate the detection and analysis of rare cells such as epithelial derived tumor cells present in the circulatory system.

  It is less clear whether the smaller the target cell population, the more difficult it is to magnetically label and recover. Furthermore, labeling and recovery are clearly dependent on the nature of the magnetic particles used. For example, when cells are incubated with large magnetic particles such as Dynal beads, the beads are too large for effective diffusion, and the cells are labeled by collisions caused by the mixing of the system. Thus, if cells that are tumor cells in very early cancers are present in the population at a frequency of 1 cell per ml of blood or less, the probability of labeling the target cell is added to the system. Related to the number of magnetic particles and the length of the mixing time. Since the cells are mixed with such particles for a considerable amount of time, it is necessary to increase the particle concentration as much as possible. However, separation can replace rare cells mixed with other blood cells with rare cells mixed with a large amount of magnetic particles, but there is a limit to the amount of magnetic particles that can be added. The latter condition does not significantly improve the ability to count the cells of interest or to examine them.

  Based on the above, high-gradient magnetic separation on external magnetic field devices using high magnetic, low non-specific binding, colloidal magnetic particles, in particular, includes a small subset of the cell subset of interest in the entire population If this is the case, then this is the method that should be selected to separate the cell subset of interest from the mixed population of eukaryotic cells. Such substances easily discover rare events such as tumor cells in the blood and magnetically label due to their own diffusion properties. For magnetic separation to make tumor cell analysis successful, the magnetic particles should be specific for epitopes that are not present on hematopoietic cells.

SUMMARY OF THE INVENTION Once a tumor cell is identified as circulating, it is desirable to further characterize the phenotypically or biochemically isolated cell. Thus, specific tumor constitution related molecules such as nucleic acids, proteins or hydrocarbons associated with a malignant phenotype can be analyzed. In particular, it is clinical in determining the type of cancer and evaluating the efficiency of chemotherapy-mediated policies by measuring the expression level of certain tumor constitution-related molecules present in or on tumor cells identified in the circulatory system Provide a way to assist the physician.

  In a preferred embodiment of the invention, a method is provided for assessing a patient for the presence of malignancy. This method involves obtaining a biological specimen from a patient containing a mixed cell population suspected of containing hematopoietic and non-hematopoietic malignant cells. A sample is then prepared in which the biological specimen is mixed with a detectably labeled ligand that reacts specifically with the malignant cells, substantially excluding other sample components. The sample is contacted with at least one reagent that also specifically labels malignant cells. The sample is then analyzed to determine the presence and number of labeled cells. Cell detection indicates the presence of malignancy, the more labeled cells, the higher the severity of malignancy. The method further includes evaluating the labeled cell for changes in at least one tumor constitution associated molecule. In one embodiment, the assessment includes contacting the molecule with a detectably labeled agent that has binding affinity for it. Tumor constitution-related molecules may be proteins, nucleic acids or hydrocarbons and are evaluated using conventional methods.

One aspect of this method is multi-parameter flow cytometry, immunofluorescence microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectroscopic image analysis, manual cell analysis (spectral imaging analysis manual cell analysis) , cell spotter ^{R (cell} spotter ^R) analysis, by a method selected from the cell Tracks (cell Tracks) the group consisting of analysis and automated cell analysis, to analyze the malignant cells.

  The method of the present invention can be used to evaluate circulating cancerous cells after medical, radiation or surgical treatment to eradicate tumors. This method can also be performed periodically over the course of several years to assess patients for the presence or number of circulating tumor cells and changes in tumor constitutional molecules as an indicator of disease onset, recurrence and / or progression. it can.

  In yet another embodiment of the invention, a method is provided for determining a change in a tumor constitution related molecule as a means for predicting a therapeutic effect. A typical method is to take a sample from the patient; if present, isolate and count circulating malignant cells from the cells; then, as a means to predict the therapeutic effect, Determining the number of at least one predetermined tumor constitution related molecule on the cell. Such methods can also be used to facilitate the evaluation of an appropriate dose for a treatment strategy and / or to monitor the effect of treatment over time. Thus, the method of the present invention provides a “whole body” biopsy based on a simple blood test.

  In yet another embodiment of the invention, a method for culturing tumor cells isolated from the circulatory system is provided. Such cells are then contacted with a therapeutic agent to assess their sensitivity to it. Such cells also provide the origin of tumor constitution related molecules that may or may not change. Thus, the present invention provides a tumor cell or culture thereof isolated from the circulatory system.

  In a preferred embodiment of the present invention, a tumor vaccine derived from the isolated circulating tumor cells of the present invention is disclosed. Such tumor vaccines will contain circulating tumor cells, fragments thereof or purified tumor constitution related molecules.

  In yet another embodiment of the present invention, a method for identifying changes in circulating tumor cells relative to cells present in an in situ tumor mass is provided. A typical method is to take a biopsy specimen of such a tumor mass from a patient and, if any are present, isolate circulating tumor cells from the patient. The specimen and isolated circulating tumor cells are then contacted with two panels of agents that detect multiple tumor constitution related molecules. Such agents are detectably labeled as desired. The circulating tumor cells and any tumor constitution related cells present in the specimen are then analyzed to determine whether the tumor constitution related molecules are altered in the circulating tumor cells relative to the biopsy specimen.

In a further aspect of the invention, a kit for screening patient samples for the presence of non-hematopoietic malignant cells is provided. A representative kit of the invention comprises (i) a magnetic core material, a protein-based coating material, and a coated magnetic nanoparticle comprising an antibody that specifically binds to the first characteristic determinant of the malignant cell, wherein: The antibody is coupled directly or indirectly to the base coating material; (ii) at least one antibody having binding specificity for a second unique determinant of the malignant cell; (iii) the malignant cell A cell-specific dye for excluding other sample components from the analysis; (iv) a device selected from the group consisting of a Cell Spotter ^R cartridge or Celltrax cartridge; and at least one having binding affinity for a tumor constitutional molecule A detectably labeled agent. Such kits optionally include an antibody having binding affinity for unlabeled cells, a biological buffer, a permeabilization buffer, a protocol, and optionally an information sheet.

DETAILED DESCRIPTION OF THE INVENTION According to a preferred embodiment, the present invention provides compositions, methods and kits for the rapid and effective isolation of rare target organisms from biological samples. The described methods can be used effectively to isolate and characterize tumor cells present in a blood sample, while minimizing the selection of non-specifically bound or captured cells.

  The cancer staging system portrays how far the cancer spreads anatomically and attempts to place patients with similar prognosis and treatment in the same staging group. The staging concept is applicable to almost all cancers except most forms of leukemia. Because leukemia is involved in all of the blood, it does not localize anatomically like other cancers, and as a result, the concept of stage is often not available for this type of cancer. Some forms of leukemia have a staging system that reflects various measures of how the disease is progressing. For most solid tumors, there are two related staging systems, Overall Stage Grouping and the TNM system.

  In a comprehensive staging (Roman numeral staging) system, the disease states are grouped into four stages, represented by the Roman numerals I-IV, or classified as “relapse”. In general, stage I, an early stage cancer, is a small localized cancer that can usually be treated, while stage IV usually represents inoperable or metastatic cancer. Stage II and III cancers usually progress locally and / or are local lymph node metastases. In fact, these staging are strictly defined, but the definition is different for each type of cancer. In addition, it is important to recognize that the prognosis for a given stage also depends on what type of cancer it is, so stage II non-small cell lung cancer has a different prognosis from stage II cervical cancer. .

  As noted above, it is common for cancer to recur months or years after the primary tumor is removed. This is because when the primary tumor is discovered, the cancer cells are already located far away, but a tumor that is not large enough to be detected at that point is formed. Sometimes a small piece of the primary tumor remains in the first surgery, which later grows into a macroscopic tumor. Cancer that recurs after eradicating all visible tumors is called a recurrent disease. Disease that recurs in the area of the primary tumor is localized recurrence, and disease that recurs as metastasis is called distant recurrence. Distant recurrence is treated in the same way as normal stage IV disease (sometimes these terms are used interchangeably). The severity of localized recurrence is quite different from remote recurrence and will depend on the type of cancer.

  For solid tumors, stages I-IV are actually defined based on a more detailed staging system called the “TNM” system. In this system, TNM means primary tumor (Tumor), regional lymph nodes (Nodes), and distant metastases (Metastases). Each of these is categorized separately and classified by number to give a total stage. Thus, T1N1M0 cancer means that the patient has a T1 primary tumor, N1 lymph node metastasis, and no distant metastasis. Of course, the definitions of T, N, and M are specific to each cancer, but their meaning can be broadly defined.

  T: Tumor—Classifies the extent of the primary tumor and is usually expressed as T0 to T4. T0 means a tumor that has not yet begun to invade local cells. This is called “In Situ”. On the other hand, T4 refers to a large primary tumor that invades other organs, possibly by direct invasion, and is not usually capable of surgery.

  N: Lymph node-classifies the amount of regional lymph node metastasis. Distant lymph node metastasis is considered a metastatic disease. The definition of exactly which lymph nodes are localized depends on the type of cancer. N0 means no lymph node metastasis, while N4 means extensive metastasis. In general, the wider the metastasis, the larger the node that has metastasized, meaning that the node has metastasized more distantly (although it is still localized).

  M: Distant metastasis-M is either M0 if there is no metastasis or M1 if there is metastasis. As with overall staging, the exact definitions for T and N vary with each different cancer type.

  Most oncologists consider the TNM system to be more rigorous than the I to IV system because it gives a more rigorous category. In practice, however, these two systems are related. The I to IV classification is actually defined using the TNM system. For example, Stage II non-small cell lung cancer is a T1 or T2 primary tumor, with N1 lymph node metastases, meaning no metastases (M0).

  Many clinicians believe that cancer is an organ-confined disease in its early stages. Based on the data presented here, this idea is wrong. In fact, the data reveal that cancer is often a systemic disease when first detected using currently available methods. Therefore, the presence of tumor cells in the circulatory system can be used in place of or in conjunction with other tests such as mammography or PSA measurements to screen for cancer. Organ origin of such cells by using appropriate monoclonal antibodies directed to relevant markers on or in target cells, or using other assays for cellular protein expression, or by analysis of intracellular mRNA For example, breast cancer, prostate cancer, colon cancer, lung cancer, ovarian cancer, or other non-hematopoietic cell cancer can be readily determined. Thus, if cancer cells can be detected but there are virtually no clinical signs of the tumor, it is possible to identify their presence as well as the organ of origin. Because screening can be performed with the relatively simple, highly sensitive and specific blood tests of the invention described herein, the test is considered a “whole body biopsy”. Furthermore, based on the data described herein, cancer is characterized by the presence of potentially highly harmful metastatic cells and is therefore considered a blood-borne disease to be treated. For example, if there is no detectable evidence of absolutely circulating tumor cells after surgery, it may be possible to determine from further clinical experiments whether follow-up treatment such as radiation, hormonal therapy or chemotherapy is necessary . It is important and useful clinical information that a patient needs such treatment or predicts its effect, given the cost of such therapy.

  From the data herein, it is also clear that the number of tumor cells in the circulatory system is associated with the progression stage from the onset to the final phase of the disease.

  The term “target organism” as used herein refers to a wide range of substances of biological or medical interest and can be distinguished from “non-target” substances present in the specimen. For example, hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemicals, nucleic acid molecules (eg, RNA and / or DNA) and particulate analytes of biological origin including biological particles such as cells, viruses, bacteria, etc. . In a preferred embodiment of the invention, the compositions, methods and kits of the invention are used to efficiently remove rare cells such as maternal circulating fetal cells or circulating cancer cells from non-target cells and / or other organisms. Can be isolated.

  The term “biological specimen” includes, but is not limited to, peripheral blood, tissue homogenate, nipple aspirate, colon lavage, sputum, bronchial lavage, and any other cell available from a human subject. Includes cell-containing body fluids including, without limitation, origin. Representative tissue homogenates are available from symptomatic lymph nodes of breast cancer patients. The term “antigenic determinant”, when used to refer to any of the target organisms, refers broadly to a chemical mosaic present on a macromolecular antigen that often generates an immune response. Antigenic determinants can also be used interchangeably with “epitope”. An antigenic determinant can be specifically bound by a biospecific ligand or biospecific reagent and participates in and selectively binds to a specific binding substance (such as a ligand or reagent) A part, the presence of which is necessary for selective binding to occur. In basic terms, an antigenic determinant is a molecular contact region on a target organism that is recognized by agents, ligands and / or reagents having binding affinity for it in a specific binding reaction.

  The term “specific binding pair” as used herein refers to antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-hydrocarbon, nucleic acid (RNA or DNA) hybridizing sequence. Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin and virus-receptor interactions. The term “gene-specific probing” refers to a method of detecting the presence or absence of such a molecule using a nucleic acid molecule that is complementary to a tumor constitution associated molecule. Such nucleic acids may or may not be detectably labeled. A variety of other antigenic determinant-specific binding agent combinations are contemplated for use in practicing the methods of the invention and will be apparent to those skilled in the art. The term “tumor constitution” as used herein refers to a predisposition or predisposition to malignancy. Susceptibility or predisposition to malignant disease may be inherited or may be due to somatic mutation that results in dysregulated cell proliferation. The phrase “tumor constitution associated molecule” refers to an intracellular or extracellular molecule that changes biochemically or is abnormally expressed when a cell progresses from a normal to a malignant phenotype. Such molecules include, but are not limited to, hormones, hormone related proteins, oncogenes, tumor suppressor proteins, apoptosis related molecules, cell cycle and proliferation related molecules, malignantly related hydrocarbon molecules, cytoskeletal proteins and Contains proteins involved in maintaining cell-cell contact. Methods for analyzing tumor constitutional molecules including proteins, nucleic acids and hydrocarbons can be found in [Current Protocols in Molecular Biology, F. M. Ausubel et al. Eds. John Wiley & Sons, Inc. NY, NY (1999)]. Assessment of altered expression levels or altered molecular structure of tumor constitution related molecules provides the clinician with valuable information to aid in the design and monitoring strategy of treatment.

  The term “malignant cell” refers to a cell that has changed biochemically and / or phenotypically and has lost normal stringent control of cell growth and / or localization. Malignant cells are usually absent from the circulatory system.

  The term “antibody” as used herein includes immunoglobulins, monoclonal or polyclonal antibodies, immunoreactive immunoglobulin fragments such as F (ab), and single chain antibodies (sfV). In the present invention, it is also contemplated to use peptides, oligonucleotides or combinations thereof that specifically recognize antigenic determinants with the same specificity as traditionally produced antibodies. As mentioned above, complementary nucleic acids are encompassed within the meaning of “specific binding pair”. The term “detectable label” as used herein refers to the presence of a target organism in the test sample, either directly or indirectly by physical or chemical means. Any substance that can be an indicator of Representative examples of useful detectable labels include, but are not limited to, the following: molecules that can be detected directly or indirectly based on absorbance, fluorescence, diffraction, light scattering, phosphorescence or luminescence properties Ions; molecules or ions detectable by their radiative properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. For example, among the group of molecules that can be indirectly detected based on absorbance or fluorescence, various molecules that yield suitable substrates that convert, for example, from non-absorbing to absorbing molecules or from non-fluorescent to fluorescent molecules. Enzymes are included.

  The phrase “for substantial exclusion” refers to the specificity of the binding reaction between the biospecific ligand or biospecific reagent and its corresponding target antigenic determinant. Biospecific ligands and reagents have specific binding activity for their target antigenic determinants, but also exhibit low levels of non-specific binding to other sample components.

The phrase “early stage cancer” is used herein to refer to those cells that have been used interchangeably with “Stage I” or “Stage II” cancer and clinically determined to be organ-localized. Also included are tumors that are too small to be detected by routine use, such as mammography for breast cancer patients or x-rays for lung cancer patients, although mammography can detect tumors with about 2 × 10 ⁸ cells, but the present invention is about this size. Allows detection of circulating cancer cells from the following tumors:

  The term “enrichment”, as used herein, means that the ratio of target biological matter (eg, tumor cells) to non-target material in a treated analytical sample is compared to the ratio in the original biological sample. Is a method that increases in number. When peripheral blood is used as a starting material, red blood cells are not counted when assessing the degree of enrichment. Using the method of the invention, circulating epithelial cells can be enriched to at least 2,500 times, more preferably 5,000 times and most preferably 10,000 times the leukocytes.

  The phrase “clonal expansion” when used in reference to isolated circulating tumor cells refers to a method of culturing the isolated cells under conditions in which the cells grow. Single cells can be cultured to form colonies, which can then be clonally expanded to produce substantially homogeneous cancer cells. Tumor vaccines can be made using portions of such cells or the cells themselves. The phrase “tumor vaccine” as used herein refers to an agent containing a tumor cell specific protein that can be used to stimulate an immune response. Vaccines can contain viruses, small proteins or whole cells. A method for making tumor vaccines using tumor cells transfected with adenovirus-related vectors is disclosed in US Pat. No. 6,171,597. A further method for generating tumor cells from circulating tumor cells is disclosed in US Pat. No. 5,993,829.

  Preferred magnetic particles or ferrofluids used to practice the present invention are particles that behave as colloids. Such particles are generally characterized by their submicron particle size, which is less than about 200 nanometer nm (0.20 microns), and their stability against gravity separation from extended time solutions. In addition to numerous other advantages, this size range is essentially invisible to analytical techniques that are commonly applied to cell analysis. Particles having a magnetic quantity in the range of 90-150 nm and between 70-90% are considered to be used in the present invention. Suitable magnetic particles consist of a crystalline core of supermagnetic material surrounded by molecules that bind, eg physically adsorb, or covalently bond to the magnetic core and stabilize the colloidal properties. The coating material should preferably be applied in an amount effective to prevent non-specific interactions between the biological macromolecules found in the sample and the magnetic core. Such biological macromolecules can include carbohydrates such as sialic acid residues on the surface of non-target cells, lectins, glycoproteins and other membrane components. In addition, the material should contain as high a magnetic quantity / nanoparticle as possible. The size of the magnetic crystals including the core is small enough that they do not contain complete magnetic domains. The size of the nanoparticles is small enough so that their Brownian kinetic energy exceeds their magnetic moment. Thus, the N-pole, S-pole alignment and subsequent mutual attractive / repulsive forces of these colloidal magnetic particles do not appear to occur even in moderately strong magnetic fields, contributing to their solution stability. Finally, the magnetic particles will be separable in a high magnetic gradient external field separator. Its properties facilitate sample processing and provide an economic advantage over more complex internal gradient columns loaded with ferromagnetic beads or cotton wool. Magnetic particles having the above properties can be prepared by modification of the base materials described in US Pat. Nos. 4,795,698, 5,597,531, and 5,698,271. Their preparation from their base materials is described below.

Malignant tumors are characterized by their ability to invade adjacent tissues. In general, tumors with a diameter of 1 mm are vascularized and animal studies show that as much as 4% of the cells present in the tumor can flow into the circulation in a 24 hour period (Butler, TP & Gullino PM, 1975). Cancer Research 35: 512-516). The ability of a tumor to flow is most likely dependent on the aggressiveness of the tumor. The volume that the tumor flows out is most likely dependent on the aggressiveness of the tumor. It is believed that tumor cells are continuously circulated into the circulation, but only a small fraction or only a small fraction will rise to distant metastasis (Butler and Gullino, supra). Using the following assumptions, the frequency of tumor cells in circulation can be estimated as follows:
1. A tumor with a diameter of 1 mm contains 10 ⁷ cells and 4% or 4 × 10 ⁵ cells will be circulated into the circulation in a 24 hour period;
2. Tumor cells persist in only one circulation cycle;
3. blood volume of about 5 liters; and 4. cardiac output of 5000 ml / min.

  In such a case, the frequency of tumor cells in the peripheral blood of a patient with a 1 mm diameter tumor is approximately 6 tumor cells / 100 ml blood. The increase in tumor mass can be expected to be proportional to the increase in the frequency of circulating tumor cells. If this proves to be the case, methods available with this level of sensitivity will facilitate assessment of tumor burden in patients with distant metastases as well as those with local disease. Detection of tumor cells in the peripheral blood of patients with local disease has the potential to not only detect tumors at an early stage, but also provide an indication of the potential invasiveness of the tumor.

  Several studies have reported the presence of cancer cells in leukopheresis products harvested from patients with breast cancer for autologous peripheral blood stem cell transplantation (Brugger W et al. (1994) Blood 83. : 636-640; Brockstein BE et al. (1996) J of Hematotherapy 5: 617; Ross AA et al. (1993) Blood 82: 2605; Ross AA. (1998) J of Hematotherapy. 7: 9-18; Moss TJ et al. ) J. Hematotherapy. 3: 163-163). These findings prompted the criticism of the use of this procedure for autologous transplantation because tumor cells in the transplant product have the potential to establish metastases. In addition, it has been found that leukocyte removal products are more likely to contain tumor cells when obtained from individuals with disseminated disease (Brugger et al., 1994, supra). However, these studies do not report quantitative data nor report that they can find tumor cells in the peripheral blood of patients with local disease. With these observations, it can be assumed that the actual tumor burden can be measured using a highly sensitive and quantitative test that counts the number of tumor cells in the peripheral blood. In order to assess the feasibility of such a test, a sensitive cell assay has been developed that allows accurate counting of circulating cancer cells limited only by the volume of blood to be tested.

It should be noted that many different cell analysis platforms can be used to identify and enumerate cells in the enriched sample. An example of such an analysis platform is the manual observation of the cells described in Example II, described in US Pat. Nos. 5,876,593; 5,985,153 and 6,136,182, respectively. Imunicon's Cell Spotter ^R system, a magnetic cell immobilization and analysis system with microscopic detection, and a newer automatic optical scanning system, Celltrax system. All of the aforementioned US patent applications disclosing manual or automated quantitative and qualitative cell analysis are hereby incorporated by reference. Such devices can also be used to benefit the diagnostic and monitoring kits of the present invention.

  Other analytical platforms include laser scanning cytometry (Compucyte), bright field image analysis (Chromavision) and capillary volumetric analysis (Biometric Imaging).

Counting circulating epithelial cells in blood using the methods and compositions of the preferred embodiments of the present invention is a sample by multi-parameter flow cytometry following immunomagnetic selection (enrichment) of epithelial cells from blood. Achieved through analysis of Immunomagnetic sample preparation, reduces the amount of sample, it is important to obtain 10 ⁴ fold enrichment of the target (epithelial) cells. Reagents used for multi-parameter flow cytometry analysis are optimized to be located at a unique location in a multi-dimensional space created by two light scattering and three fluorescence parameter listmode collections. These include the following:
1) An antibody against CD45, a pan-leukocyte antigen for identifying leukocytes (non-tumor cells);
2) cell type-specific or nucleic acid dyes that allow the elimination of residual red blood cell, platelet and other nuclear-free events; and 3) biologically specific reagents or antibodies directed against cytokeratin Or an antibody having specificity for an EpCAM epitope different from that used to immunomagnetically select cells.

  It will be appreciated by those skilled in the art that the method of analysis of the enriched tumor cell population will depend on the intended use of the invention. For example, as described below, the number of circulating epithelial cells can be very low in cancer screening or monitoring for disease recurrence. Analytical methods that identify epithelial cells as normal or tumor cells are desirable because there are some “normal” levels of epithelial cells (though very likely when introduced during venipuncture). In that case, a microscope-based analysis can be found to be the most accurate. Such testing may also include morphological assessment and identification of known tumor constitution related molecules (eg, oncogenes). Suitable tumor constitution related molecules that can be further analyzed according to the method of the present invention are provided in Example 11.

  Alternatively, analytical methods that count such cells in disease states where the number of circulating epithelial cells far exceeds that observed in the normal population would be sufficient. Determination of patient status by the methods described herein is made based on a statistical average of the number of circulating rare cells present in a normal population. In addition, as described herein, circulating epithelial cell levels in early stage cancer patients and patients with aggressive metastatic cancer can also be statistically determined.

  The following examples are provided to facilitate the practice of the present invention. These examples are not intended to limit the scope of the invention in any way.

Example 1
Improved magnetic nanoparticle formation for effective isolation of rare cells from whole blood Rare cells (eg, tumors in patients with epithelial derived tumors, fetal cells in maternal blood, etc.) 1 ml It can be present at a frequency of less than 1 rare cell per blood. The number of blood smears required to detect such rare cells is very large. Assuming 10 rare cells in 10 ml of blood, it corresponds to 10 tumor cells in 5-10 × 10 ⁷ white blood cells (white blood cells) and the cells are either cytocentrifugation or established. (Settling) can be transferred to a microscope slide, stained with an antibody specific for the rare cell of interest, and read manually or automatically. The maximum number of cells that can be transferred to one slide is approximately 500,000 cells, meaning that 100-200 slides are required to process 10 ml of blood. The time required for analysis with this approach makes it impractical and difficult to implement economically. As a result, enrichment methods such as sample volume reduction and red blood cell and platelet removal by density gradient separation or red blood cell lysis procedures are used to isolate rare cells and significantly reduce the number of slides to be analyzed. As noted above, magnetic enrichment is a preferred method for cell separation, and ideally the nanoparticles used for this purpose do not need to be removed prior to analysis. Thus, the nanoparticles will be small enough so as not to interfere with analytical measurements, i.e., less than about 250 nm. More preferably, the nanoparticles are less than 220 nm so that they can be filter sterilized. Furthermore, the nanoparticles are large enough and sufficiently magnetically responsive to allow cell separation from a single experimental test tube in an external gradient magnetic separator, i.e., test tube, centrifuge tube, vacutainer, etc. Should be. Again, as noted above, it is noted that the internal gradient device is cumbersome, expensive and inefficient in collecting rare cells. Nanoparticles and magnetic devices should also provide high and reproducible recoveries with low non-specific binding. U.S. Pat. No. 5,597,531 describes the synthesis of advanced magnetic particles called directly coated (DC) particles having many of these properties. These nanoparticles are composed of a spherical mass of crystalline magnetite or other magnetic oxide in appearance, which is coated with a polymer or protein (based on the coated magnetic particles). Because of their structure (core and polymer coatings where the core diameter is >> from the thickness of the coating), they are about 80-85% magnetic, non-specific binding of these nanoparticles is 5 They are in the range of -8%, so they are not very practical for rare cell separation. Thus, if one cell per ml and enriching the cells present at 80% capture efficiency, the best result expected with 10 ml whole blood (think white blood cells only) is 4 million. There will be 8 cells recovered in total, ie 16-17 fold enrichment. However, the magnetic particles described in US Pat. No. 5,597,531 have suitable magnetic properties for performing separation from a single test tube using an open field separator. Furthermore, their average size is sufficient under the limits suggested above, so they do not interfere with various analytical procedures. Based on expensive testing with those materials, the major contributor to non-specific binding to cells was found to be the presence of exposed crystalline iron oxide on the nanoparticles due to imperfect coating It was done. Such imperfectly coated crystals have a sufficiently high positive charge at physiological pH where they appear to bind strongly and very strongly to biological macromolecules such as negatively charged sialic acid on the cell surface. ing. An improved method for making particles is described in US Pat. No. 5,698,271. These materials are an improvement over those disclosed in the> 531 patent in that the process includes a high temperature coating step that significantly increases the level of coating. For example, using this process, nanoparticles made with bovine serum albumin (BSA) coatings are 3-5 times lower in cells when compared to the DC-BSA material of US Pat. No. 5,579,531. Has specific binding properties. This decrease in non-specific binding has been shown to be directly due to increased levels of BSA coating material. When such nanoparticles are treated to remove the BSA coating, non-specific binding returns to a high level. Thus, it was determined that there was a direct correlation between the amount of BSA covered on the iron oxide crystal surface and nonspecific binding of cells. Typically, non-specific binding of these particles to cells from whole blood was 0.3%, which was much better than that generated from US Pat. No. 5,579,531. Thus, from 10 ml whole blood there will be about 200,000 non-target cells that are also isolated with cells targeted for enrichment.

  In addition to the non-specific binding problem to be addressed below, different lots of magnetic particles made as described in US Pat. Nos. 5,579,531 and 5,698,271 can be Used for depletion or enrichment, recovery was not consistent. Sometimes recovery was 85-95%, others could be 40-50% using the same model system. Since the manufacturing methods of these materials produce a substantial range of size dispersion (30 nm to 220 nm), it was speculated and confirmed that the size distribution and especially the presence of small nanoparticles significantly affects the target recovery. As small nanoparticles (30-70 nm) diffuse more easily, they will preferentially label cells compared to their larger counterparts. When using very high gradients, such as internal gradient columns, the efficiency of these materials was little different regardless of size. On the other hand, the occupancy of small nanoparticles on cells has an important effect when using external gradients, or a smaller degree of gradient that can occur on microbead or steel wire columns. This was finally shown to be the case by fractionating DC nanoparticles and studying the impact on recovery. Based on these studies and other optimization experiments, when base coated magnetic particles can be prepared, a means to fractionate nanoparticles magnetically or on a column is established, which is excessively small or large It lacked nanoparticles. For example, base coated particles with an average diameter of 100 nm can be produced, which best contains trace amounts of material less than 80 nm or greater than 130 nm. Similarly, materials of about 120 nm could be made, and materials below 90-95 nm and above 160 nm were not significant. Such materials were optimized for recovery and could be somewhat optimized by including 60-70 nm nanoparticles. A preferred particle size range for use in the practice of the present invention is 90-150 nm for magnetic particles coated with a base, eg, magnetite coated with BSA. Particles within this preferred range can be obtained using the procedure described by Liberti et al. In Fine Particles Science and Technology, 777-90, E. Pelizzetti (eds.) (1996).

  In order to further address the non-specific binding problem, several routes were attempted to create nanoparticles directly conjugated with antibodies. Monoclonal antibodies specific for rare cells can be coupled directly to a BSA base coated on DC magnetic particles by, for example, standard heterobifunctional chemistry (herein, direct coupling). The law). Heterogeneous bifunctional linkers used for these purposes include sulfosuccinimidyl-4- [maleimidomethyl] cyclohexane-1-carboxylate (SMCC). In another approach, biotinylated monoclonal antibodies can be coupled to streptavidin coupled to base-coated particles. This conjugation method is referred to in the specification as a piggyback method. In this process, streptavidin is coupled to magnetic particles whose base is covered by the same chemistry as the direct coupling method. In one type of piggyback coupling method, a monobiotinylated antibody is reacted with streptavidin magnetic particles for 1 hour and then the remaining streptavidin binding sites are suppressed with free biotin. It is important to suppress the streptavidin sites remaining after antibody coupling to prevent binding of any biotinylation step to magnetic particles during rare cell isolation or cell analysis steps. Furthermore, this means for suppressing streptavidin has been shown to be effective in combating non-specific binding. Incubation of such materials under various conditions with biotinylated fluorescent macromolecules results in no bound fluorescence. For comparison, an anti-EpCAM antibody (GA73.3 obtained from Wistar Institute, Philadelphia, Pa) was coupled to magnetic particles by both methods. Both magnetic particles were then compared for selection of cells from a colon tumor cell line (Colo-205) added to whole blood as well as leukocyte non-specific binding (NSB) or carryover. The leukocytes present in the final sample were a combination of leukocytes non-specifically bound to magnetic particles and cell carryover from the washing step. It is noted that following magnetic selection, it is necessary to wash any cells that were in contact with the test tube at the start of the separation or that were non-magnetically transferred during the separation process. Table I shows a comparison of these two magnetic particles.

  The first to note is that simple coupling of antibody or streptavidin to BSA-based particles significantly reduces non-specific binding (data not shown). This is believed to reduce the accessibility of the “exposed” crystal surface to the cell per bond. The above table shows that the recovery of added cells is comparable for both types of magnetic particles. However, nonspecific binding of leukocytes was three times higher when using magnetic particles directly coupled with antibodies. This difference is important when large amounts of blood are processed and analyzed, albeit relatively small. A reasonable explanation based on numerous supporting observations on the difference between the two types of magnetic particles is that there are more layers of protein on the magnetic particles synthesized using the piggyback coupling method. is there. Thus, it appears that the surface of the magnetic crystal is more extensively covered with multiple layers of protein and is “sterically” protected. This prevents non-target cell binding to magnetic particles.

  In the piggyback coupling method, a limited number of streptavidin binding sites on the magnetic particles are coupled with biotin-antibody and the rest is saturated with free biotin by the aforementioned inhibition process. In yet another coupling method, excess streptavidin binding sites were suppressed and saturated with monobiotin-BSA instead of free biotin. The rationale for this approach is that inhibition with monobiotin BSA further sterically inhibits the cells that come into contact with the uncoated areas of the nanoparticles, i.e. gives a good coverage of the nanoparticles. Carbon analysis has shown that this process increases the amount of protein coupled to the particles. The two magnetic particle preparations were compared in experiments evaluating recovery of added Colo205 from whole blood, or nonspecific binding of leukocytes. The results are shown in Table II.

  Monobiotin-BSA can be prepared by conjugating a limited amount of biotin to BSA so that 30-40% of the resulting product does not bind to biotin.

  In summary, magnetic particles with a homogeneous size distribution and streptavidin binding sites suppressed with biotin BSA perform very well in the assay method of the present invention. Good recovery of added epithelial tumor cells and an order of magnitude reduction in non-specific binding were obtained using these particles compared to nanoparticles blocked with biotin. Thus, these materials and the results obtained with them will define very useful products that can be further optimized. The improved ferrofluid product is as magnetic as possible and coated to eliminate all possible interactions between its magnetic core and any substance in the blood, including cells. Coated with a polar monolayer) and is well defined in its size range and distribution. In a preferred situation, a coating material that does not interact with biological material is used. Where such interactions are unavoidable, means are needed to block them. For materials where the material should be as magnetic as possible, those described in US Pat. Nos. 5,579,531 and 5,698,271 are preferred starting materials. They are preferred because they are not perfect, but are composed of a large magnetic core with an apparently single layer base coating material. For 100 nm nanoparticles coated with BSA, the core would be a suitable magnetic oxide such as about 90 nm magnetite. Due to the relative size of the core and the coating material, such nanoparticles are as clearly magnetic as possible. This is believed to be apparent if the function of the coating keeps the nanoparticles interacting undesirably with each other, which will lead to macroscopic agglomeration. The coating also promotes sufficient interaction with solvent molecules to maintain colloidal behavior and provides a convenient chemical means for coupling. Also, the nanoparticles of US Pat. Nos. 5,579,531 and 5,698,271 are filled with “holes” in a monolayer, ie sterically physically, in several ways. It is preferred as a starting material because it has a sufficiently single layer coating. Obviously, any coating that promotes effective and complete coverage that would inhibit the interaction of the core material with blood components or any other non-specific effect in any other system would be suitable. Let's go. The fewer coatings that can be added to the nanoparticles, the better, so as to maximize the magnetic quantity-to-nanoparticle quantity ratio.

Example 2
Semi-automated sample preparation and analysis for identification of circulating tumor cells A semi-automated system was developed to process and analyze 7.5 ml of blood for the presence of epithelial derived tumor cells. Cells of epithelial cell origin are immunomagnetically labeled and separated from the blood. Magnetically captured cells are labeled with different fluorescence and placed in the analysis chamber. Four-color fluorescence images are used to distinguish between epithelial origin residues, hematopoietic cells and circulating tumor cells (CTC). An algorithm is applied to the captured images to count internal controls and identify all subjects that potentially classify as tumor cells based on size and immunophenotype. A thumbnail image of each subject is shown in the user interface, from which the user can determine the presence of tumor cells. In normal donor blood processing, the internal controls showed consistent and reproducible results between the system and the operator. CTCs were detected in blood samples of patients with metastatic breast cancer, however other diseases can be analyzed with the system.

  As noted above, tumor cells can be present in the blood of cancer patients at a very low frequency (<10 cells per ml). The cumbersome manual sample preparation involved in detecting the presence of CTC and complex analytical methods often leads to erroneous results. For highly complex experimental procedures, the root cause of false results is often systematic and / or random preanalytical errors, i.e. in the sample preparation or pretreatment stage rather than in the analytical procedure itself. You can trace the cumulative effects of errors that occur in between. Pre-analytical errors can be shown as variations due to method-sensitive process steps as well as random or systematic variations for each operator. Thus, manual sample preparation in rare cell analysis can result in high variability and unreliable analytical results if done inconsistently. Thus, automation of such a pre-analysis process minimizes variability and provides a more consistent analysis result. An analytical technique frequently used for analyzing the prepared sample is flow cytometry or microscopy. Although flow cytometry is sensitive and reproducible, researchers cannot confirm that rare events that are immunophenotypically identified actually display morphological features consistent with tumor cells . Although microscopy increases confidence in determining malignancy, it has the disadvantage that significant and variable cell loss is associated with sample processing. Here we introduce a novel cell presentation device and method that enables efficient collection and analysis of CTCs in a semi-automated four-color fluorescence microscope system.

  The following materials and methods are provided to facilitate the implementation of Example 2.

  The system is prepared with System Buffer (Immunicon Corp, Huntingdon Valley, PA), which is further used for various steps during the procedure. This buffer consists of phosphate buffer, EDTA, and protein to reduce non-specific binding of reagents to cells and system components. The magnetic nanoparticles are coupled to a monoclonal antibody (mabs) specific for the epithelial cell adhesion molecule (EpCAM) described in Example 1. The EpCAM antigen is expressed on cells of epithelial origin but not on cells of hematopoietic origin (Momburg et al. Cancer Research (1987) 47: 2883-2891; Gaffey et al. Am. J. Surg. Path. (1992) 16: 593-599; Herlyn et al. J. Immun. Meth. (1984) 73: 157-167; De Leij et al. Int. J. Cancer Suppl. (1994) 8: 60-63). In addition to anti-EpCAM antibody, desthiobiotin is coupled to magnetic nanoparticles to form CA-EpCAM and the reagent aggregates by adding soluble streptavidin to multiple magnetic nanoparticles in the crosslink on CTC Can be controlled, thereby increasing cellular magnetic loading and capture efficiency (Liberti et al. (2001) Journal of Magnetism and Magnetic Materials 225: 301-307). Streptavidin in AB buffer (System buffer containing added streptavidin) is added to the specimen prior to the addition of CA-EpCAM to form aggregates, which are the magnetic capture efficiency of cells at different EpCAM antigen densities Minimize the difference in. Anti-cytokeratin (CK-PE) and allophycocyanin conjugated to phycoerythrin in addition to nucleic acid-specific dye DAPI (4,6-diamidino-2-phenylindole) Fluorescent labeling with anti-CD45 conjugated to (CD45-APC). Anti-cytokeratin recognizes keratins 4, 6, 8, 10, 13, and 18 present in cells of epithelial origin. The mab is added to a buffer medium containing a surfactant to permeabilize the cell's cytoplasmic membrane. The final resuspension buffer in Cellfix (Immunicon Corp, Huntingdon Valley, PA) is phosphate buffered saline containing biotin and cell stabilizers. Biotin functions to displace desthiobiotin from streptavidin by virtue of its high affinity for streptavidin, thereby streptavidin on the ferrofluid particles in the aggregates formed in the initial steps of the assay And reverse control of cross-linking between desthiobiotin.

  To monitor the accuracy of the procedure, a known number of internal control cells are added to the blood before treatment. Internal control cells are the subject of US patent application Ser. No. 09 / 801,471, the entire disclosure of which is hereby incorporated by reference. These control cells can be successfully derived from cancer tumor cell lines. As described herein, cells SKBR-3 from the breast cancer line are stabilized with the fluorescent membrane dye DiOC16 (from Molecular Probes) and uniquely labeled to allow differentiation from endogenous tumor cells. . Approximately 1000 control cells are added to each specimen. Control cells express EpCAM and capture simultaneously with the tumor cells. Control cells also express intracellular cytokeratin and staining with CK-PE confirms the nature of this reagent. The percent recovery of added control cells provides an indicator of overall reagent and system efficiency for each sample, unlike external controls that can only detect systematic errors.

Sample Preparation System This system is equipped with two magnetic separators, each of a set of four rectangular rares arranged in a quadrupole arrangement with a 17 mm diameter hole surrounded by a circulating steel yoke. It consists of an earth magnet. In this system, each separator can hold a 15 ml conical tube. In other system arrangements, different test tubes can be used with different separators. Adjacent to each separator is a magnetic yoke that holds an analysis chamber (cell spotter ^R chamber) into which the final sample is transferred. This chamber assembly can be removed from the system and put into a microscope process. Test tube transport consists of two movable test tube arms for raising and lowering the test tube in and out of the magnetic separator, with a rotating turntable to position the test tube at various process positions. Alternatively, the probe washer is mounted on a turntable that allows the internal and external suction and transfer of the probe to be transferred. A Cavro XE1000 digital syringe pump equipped with a 5 ml syringe is used for fluid delivery delivery and probe cleaning. A Cavro SP Smart Peristaltic pump with PharMed tubing is used for aspiration or disposal. Two pinch valves (Bio-Chem) and a 3-way valve (Bio-Chem) are used to control the fluid path. A separate aspiration and transport probe made from 13 AMG Inconel tubing is used for fluid access to 15 ml conical tubes and chambers. A through-beam optoelectronic sensor (Omron) is used to determine the height of the packed red blood cell layer, allowing for precise control of plasma aspiration. The system uses an 8-bit microcontroller that tries the firmware to perform motion control, process control and operator interface instructions. The protocol itself (incubation time, fluid handling steps, etc.) is encoded into a separate memory chip. The operator interface consists of a keypad with 4 × 5 keys, a 2 × 20 character LCD display, and an audible alarm.

Cell Spotter ^R Chamber Collect the prepared sample using a 29.7 × 2.7 mm top surface smooth display screen and a molded chamber (approximately 320 μl volume) with a depth of 4 mm. 99% of the sample is placed in the chamber display screen in the chamber port and the chamber filling is sealed with a plug designed to be reproducible and efficient. In addition, a special calibration chamber having a registration mark at the center of the chamber can be used to calibrate the center of the chamber compensated from the microscope stage home position and the illumination source.

Cell Spotter ^R System The Cell Spotter ^R system is controlled by a Ludl MAC2002 controller with 10x objective lens (WD 4mm, NA 0.45), high resolution X, Y, Z stage, and this time through PC via RS-232 Utilizing a Nikon E-400 microscope equipped with a filter cube changer control controlled by Excitation, two-color and emission filters in each of the four cubes are 365 nm / 400 nm / 400 nm for DAPI, 480 nm / 495 nm / 510 nm for DiOC16, 546 nm / 560 nm / 580 nm for PE and 620 nm / 660 nm / 700 nm for APC. The images require a Hamamatsu 12-bit, 1280 × 1024 pixel digital camera connected to a National Instruments PCI-1424 frame grabber. Data collection software was developed using LabVIEW ^R and IMAQVision Toolkit ^R. The data analysis and display program was created using IBM's DB-2 database via an HTML interface.

Semi-automated sample preparation system The steps involved in the preparation of a sample for analysis are shown in FIG. To a 15 ml conical tube, add 7.5 ml blood, 6 ml system buffer and 100 μl (about 1000) control cells and mix. The sample is centrifuged at 800 g for 10 minutes and then placed in the system. The system is located on top of the packed red blood cell layer in a test tube and a probe is introduced into the test tube to aspirate plasma without disturbing the buffy coat layer. Remove the tube from the system and add 6 ml AB buffer and 100 μl CA-EpCAM ferrofluid and mix. The test tube is placed in the system, a sample test tube is inserted and retrieved from the magnetic separator under system control, thereby providing precise control over separation and removal times. In that magnetic field, the ferrofluid is moved along the side through the blood sample, thereby increasing the labeling efficiency of potential target cells, and the magnetically labeled CTC and unbound ferrofluid are removed from the test tube. Move to the wall. After 20 minutes of incubation and separation, the probe is gradually lowered into a test tube and the blood sample to be discarded is aspirated and discarded. The test tube is mechanically moved out of the separator and 3 ml of system buffer is added to the test tube. The collected cells and ferrofluid are resuspended by mixing the test tubes. The system lowers the test tube to the magnet and sucks uncollected material after 10 minutes. The test tube is moved out of the magnet by the system and 200 μl of Immunooperm and 60 μl of staining reagent are added and mixed. After 15 minutes incubation, excess staining reagent is aspirated and discarded by the system and the test tube is moved out of the magnet. Ten minutes after adding 250 μl Cellfix, the system transfers the sample into the chamber. The chamber volume is about 320 μl, and the system uses 100 μl system buffer as a rinse to ensure that cells remaining in the test tube are transferred to the chamber. The chamber is slightly overfilled to avoid air intake while capped with a plug seal. After each step in which the probe contacts the sample, the probe is thoroughly washed by the system to eliminate any cell or reagent carryover.

Cell spotter ^R analysis chamber.
FIG. 2A shows a magnet yoke assembly that holds an analysis chamber and a chamber between two magnets. To determine the optimal angle of the magnet and the optimal position of the chamber relative to the magnet formed at the two angles, a computer program was created to simulate the movement of magnetically labeled cells within the chamber. . The subject was to move all the magnetically labeled cells to the upper surface of the chamber while preventing movement to the magnetic column. Also, the distance from the chamber surface to the surface of the magnet must be short enough to allow viewing through the microscope objective. FIG. 2B shows such a simulation where the chamber is bird's-eye view between the north (N) and south (S) poles of the magnet and the dashed lines show the trajectories of the magnetically labeled cells. FIG. 2C shows an enlargement of the trajectory in the chamber. All cells labeled with magnetic nanoparticles move vertically into the chamber. FIG. 2D shows a top view of the Cell Spotter ^R chamber from an experiment in which cells and magnetic nanoparticles are introduced into the chamber. Homogeneously distributed horizontal lines on the surface of the chamber represent magnetic nanoparticles aligned along the magnetic field lines.

Data collection The chamber surface is 80.2 mm ² and must be scanned thoroughly for any object stained with DAPI, DiOC16, CK-PE and CD45-APC. The combination of object and digital camera results in a pixel size of 0.45 (0.67 × 0.67) μm ² and an image size of 858 × 686 μm. In order to cover the complete chamber surface, the Cell Spotter ^R system requires four rows of 35 images for each of the four filters yielding 140 frames and 560 images per chamber. When starting a test of a sample, the Cell Spotter ^R acquisition program is used at the area where the image is acquired, the number of images acquired for use at each position, the number of images acquired, the position of each image and each position. Automatically determines the focus of the microscope The image acquisition area is determined by moving the X and Y stages to five positions where the end of the chamber is visible in the image. The software determines the edge position and writes a line on the image display where it finds the line, giving the operator the ability to accept or invalidate the selected edge position. In addition, two measurements were taken on one of the long ends of the chamber to determine the angular offset of the chamber relative to the X axis of the stage. All images need to be in focus in all imaging areas. The depth of focus of the microscope is less than 10 μm. Although the chamber surface is planar within 10 μm, mechanical tolerances within the microscope stage, magnetic yoke and chamber can produce an oblique angle with respect to the Z axis. Due to time constraints, it is not feasible to have the software iteratively find at each of the 140 video locations on the sample chamber. An algorithm has been developed in which the software makes repeated measurements of the focus at five locations on the chamber using light emitted by nucleic acid staining of the cells. The software then gives empirical focus data a second order polynomial fit that is used to determine focus or Z adjustment at all image positions on the sample chamber. This iterative focus procedure has unique features for performing focus algorithms only on cells within a user-configurable size range and ignores non-cellular objects in the sample. In the event that no suitable particle is found, the system will move to alternate the focal point in the sample. All images from the sample are recorded in a directory that is specific to a particular sample identification.

Data analysis FIGS. 3A-3D show DAPI (panel 3A), DiOC16 (panel 3B), CK-PE (panel 3C) out of 140 frames obtained after processing a 7.5 ml blood sample from a breast cancer patient. ) And CD45-APC (panel 3D). In the DAPI image, a plurality of cell nuclei can be observed. A rectangular box is drawn around seven nuclei. The corresponding DiOC16 image shows the same seven rectangular boxes. In five of these boxes, round fluorescent objects are typically present for control cells added to the blood prior to sample processing. Control cells are also stained brightly for CK-PE, as shown by the bright staining of cells in the same 5 boxes shown in the CK-PE image. Two of the seven boxes did not stain in the DiOC16 (control) image stained in the CK-PE image. One of the boxes shows two cells and bright CK-PE staining corresponds to two cells. CD45-APC images showed no staining in the box confirming that the box contains two cells of epithelial cell origin. Other boxes showed faint CK-PE staining and bright CD45-APC staining besides this event as epithelial cells. The algorithm is applied to all images required from the sample to search for the location to stain for DAPI, DiOC16 and CK-PE. If the stained area matches that of a control cell (DiOC16 +, CK−, PE +), the software assigns this position (box) to the control cell. Data analysis software tabulates the number of control cells found in the sample. If the stained areas match those of potential tumor cells (DAPI +, DiOC16-, CK-PE +), the software saves the location of these areas in the database. The software displays a thumbnail for each box for each parameter in the row. From left to right, these thumbnails represent the nucleus (DAPI), cytoplasmic cytokeratin (CK-PE), control cells (DiOC16) and surface CD45 (CD45-APC) staining. The composite image shown on the left shows a false color overlay of nuclear (DAPI) and cytoplasmic (CK-PE) staining. A check box near the composite image and a CD45-APC box allow the user to confirm that the image represented in the column matches the tumor cells or is stained with the leukocyte marker CD45. The software creates a checked box for each sample, and that information is stored in a database. FIG. 4 shows thumbnails of six stained areas showing characteristics that are consistent with CTC from breast cancer patient samples. Three images of the six stained regions display CTC features as visualized by distinct nuclei, cytoplasmic cytokeratin staining, and absence of DiOC16 and CD45 staining. Note the difference in the appearance of the tumor cells: the cells in column 201 are relatively small, a cluster of three tumor cells is present in column 202, and some very large cells are shown in column 204. In column 203, control cells are shown at the top of the box. The area is displayed for positive CK-PE events below the control cells. However, the nuclei shown belong to leukocytes and are not consistent with CK-PE staining. Debris stained as positive in all four filters and in row 206 is shown, CK-PE positive events are not consistent with DAPI staining.

System efficiency To compare manual versus system sample preparation, a 7.5 ml aliquot of blood was added to control cells and processed in both ways. In six experiments, average tumor cell recovery was 68% for manual and 94% for system sample preparation, which had coefficients of variation of 10% and 7%, respectively. The linearity of the system is 0, 50, 100, 150 and 200 of control cells (n = 1000) and tumor cell line SKBR-3 in 7.5 ml aliquots of blood obtained from 5 normal donors. Tested by adding cells. The average recovery of control cells in these 25 experiments was 81% with a coefficient of variation of 7.8%. This indicated that the sample was processed properly. The correlation between the number of added cells and the number of recovered cells is r ² = 0.99, with a slope of 0.84 and an intercept of 3.2, 84% of tumors independent of the level of tumor cells added Cell recovery was shown. The sensitivity of the system was tested by adding 0 and 10 tumor cells to 7.5 ml aliquots of blood from 10 donors. The average control cell recovery in 20 experiments was 85% with a coefficient of variation of 7.7%. The certainty of the number actually added decreases with the number of cells added. The experiment was performed on two different days: on day 1 the coefficient of variation when 10 cells were added was 25% and on day 2 it was 15%. In samples not added, tumor cells were not found after treatment. In contrast, tumor cells were detected in all added blood samples ranging from 6-15 tumor cells (average 10.5 cells, CV 32%). The data clearly showed that the sensitivity of the system was limited only by the volume of blood processed.

  In order to characterize the efficiency of the sample preparation and analysis system between various sites and operators, six systems were placed at different sites. Blood samples from 99 healthy donors were processed in duplicate at these sites. The average recovery of control cells across the 6 sites was 77.1% with a coefficient of variation of 9.70. As expected, the reproducibility between duplicate samples is better with a coefficient of variation of 4.90. The number of events classified by the software as potential tumor cell candidates varied between 10 and 304 events, with an average of 55. An overview of candidate events is that in 28 of 192 (14.6%) blood samples, one cell was classified as a CTC membrane, and in 9 samples 2 cells (4.7%) and 3 3 cells (1.6%) in 1 sample and 13 (0.50) CTCs in 1 sample were found. Table III shows the results of these experiments for each site. In 22 patients treated for metastatic breast cancer, 16 to 703 (mean 116) candidate CTCs were found. An overview of candidate events is found in 13 out of 22 blood samples with less than 3 cells classified as CTCs, 3-10 CTCs in 4 samples, and more than 10 in 5 samples CTC was found.

  Although very rarely, circulating tumor cells can be detected in the blood of patients with adenocarcinoma. In order to investigate the potential use of CTCs in the management of cancer patients, a system that can accurately and reliably count and characterize CTCs is needed to conduct controlled clinical studies. The CTC number can represent tumor burden and changes in the CTC number can provide a means to assess the effectiveness of a given treatment. Analysis of CTCs for the presence and absence of therapeutic targets can be used to guide the treatment. The detection of CTC in individuals who are said to be healthy represents an advance in the early detection of cancer. If such early detection is possible, a non-invasive “whole body” biopsy of the solid tumor can be performed by a blood test.

To this end, a semi-automated system was developed that immunomagnetically separates epithelial cells from 7.5 ml of blood while simultaneously reducing the sample volume and immunofluorescently labeling the cells. The system creates a 320 μl liquid sample that is transferred to the analysis chamber and a magnetic device that allows all magnetically labeled cells in the sample to be drawn to the upper inner surface of the analysis chamber. Four-color fluorescence analysis is performed on samples by the Cell Spotter ^R system, counting internal control cells and potentially classifying as tumor cells by CD45 for cell surface staining nuclei, cytoplasmic positive staining and their lack. Thumbnails of all subjects that are potentially classified as used cells are displayed in the user interface where tumor cells can be finalized by the user.

  The sample preparation performed by the system is advantageous when compared to manual preparation of blood samples as indicated by higher recovery of counted tumor cells and better reproducibility. Data from the addition experiment showed the excellent linearity and sensitivity of the system. To demonstrate reproducibility, duplicate blood samples from 99 regular donors were processed at 6 different sites. Data across all sites showed a level of reproducibility as assessed by internal control cell recovery. The average recovery of the internal control was 77.1%, with a coefficient of variation varying between 3.2% and 11.8% (average 9.70). The sensitivity of the system was determined by its ability to detect CTCs in patient samples and specificity by identifying CTCs in normal donor blood. The analysis software identified between 10 and 304 candidate events (mean 55) in 192 normal blood samples, and an overview of these candidates showed an average of 0.4 events classified as CTC. . In 22 patients treated for metastatic breast cancer, 16 to 703 (mean 116) candidate CTCs were found and 0-59 (mean 7) were classified as CTCs. In the blood of 15 of 22 patients, the number of CTCs exceeded the upper limit of the 99% confidence interval for the average CTC count in normal individuals (mean + 2.6 * SE = 0.56) . In one normal blood sample, 13 events were classified as CTC by the operator, but examination of the data revealed that this could be attributed to internal control cells that were weakly stained with DiOC16. In order to avoid potential false positive results due to misclassification of control cells, the addition of internal control cells is simply done to show system and operator proficiency before trying the actual patient sample Will. Consistent and reproducible results between systems and operators in sample preparation and CTC analysis are the opportunity to conduct controlled clinical studies to elucidate the role of CTC levels in the management of patients with adenocarcinoma I will provide a.

Example 3
Counting circulating epithelial cells in patients treated for metastatic breast cancer The following method is provided to facilitate the implementation of the following examples.
patient. With informed consent, 8-20 ml blood samples were obtained from controls and patients with breast, prostate and colon cancer. Blood was obtained from several of these patients at several time points over a one year period. Blood samples were drawn into Vacutainer tubes (Becton-Dickinson) containing EDTA as an anticoagulant. Samples were kept at room temperature and processed within 24 hours after collection. Circulating epithelial cells were counted in peripheral blood samples from breast, prostate and colon cancer patients and in normal controls with no evidence of malignancy. The date of diagnosis, treatment intervention and clinical status were retrieved from the patient chart. The institutional review board of the partner organization approved the protocol.

Sample preparation.
Monoclonal antibodies specific for epithelial cell adhesion molecule (EpCAM) are extensively reactive with tissues of epithelial cell origin (Stahel RA et al. Int J Cancer Suppl. 8: 6-26 (1994); Momburg F et al. Cancer research. 47: 2883-2891 (1987); Gaffey MJ et al. Am J Surg Path. 16: 593-599 (1992)). GA73.3 or MJ37 EpCAM antibodies that recognize two different epitopes of EpCAM (kindly provided by D Herlyn (Herlyn D et al. J Immunol Methods. 73: 157-167 (1984)) Wistar Institute, Philadelphia, PA and MJ Mattes (De Leij L et al., Int J Cancer Suppl. 8: 60-63 (1993)) Center for Molecular Medicine and Immunology, NJ). 512,332 (1996), Immunicon, Huntingdon Valley, PA). The blood was incubated with anti-EpCAM conjugated ferrofluid for 15 minutes in a disposable tube with an internal diameter of 13 mm. The test tubes were placed in a separator consisting of 4 counter magnets for 10 minutes (QMS 13, Immunicon, Huntingdon Valley, PA). After separation, blood was aspirated and discarded. The test tube was removed from the magnetic separator and the collected fraction was resuspended from the vessel wall with 2 ml of FACS permeabilizing solution (BDIS, San Jose, Calif.) And placed in the magnetic separator for 5 minutes. The solution is aspirated and discarded, then the cells are resuspended in 150 μl cell buffer (PBS, 1% BSA, 50 mm EDTA, 0.1% sodium azide) and conjugated with phycoerythrin (PE). CD45 labeled with keratin (CAM5.2 monoclonal antibody) and Peridinin chlorophyll protein (PerCP) was added under saturated conditions. After 15 minutes of incubation, 2 ml of cell buffer was added and the cell suspension was then magnetically separated for 5 minutes. After discarding the unseparated suspension, the harvested cells were resuspended with 0.5 ml of its buffer and nucleic acid dye in the Procount system from BDIS, San Jose, CA was added to it according to the manufacturer's instructions. . In some cases where EpCAM antibody MJ37 was used in ferrofluid, selected epithelial cells were identified using GA73.3 PE. In these cases, cell permeabilization is not necessary. Reagents for flow cytometry were kindly provided by BDIS, San Jose, CA.

  Typical methods for determining circulating epithelial cell tissue sources use cytochemical and immunological identification techniques. Cytokeratin 5, 6, 8, 18 (CK, 5D3, LP34, Novocastra), MUC-1 glycoprotein (MUC-1 (Ma695 Novocastra)) or prostate specific antigen (PSMA), Dr. Neil Bander (Cornell University, Ithaca , NY) primary monoclonal antibody recognizing clone J591 was added to the slide after blocking non-specific binding sites with 5% BSA for 30 minutes. Samples were incubated for 20 minutes at room temperature, washed twice with PBS for 5 minutes, and then exposed to a second rabbit anti-mouse Ig (Z0259, Dako Corp., Carpenteria, Calif.) For an additional 20 minutes. After two additional washes, the samples were incubated with alkaline phosphatase anti-alkaline phosphatase (APAAP) rabbit Ig complex for 15 minutes. Finally, the addition of enzyme substrate ((New Fuchsin, Dako Corp. CA) resulted in the formation of a red precipitate whose nuclei were counterstained with hematoxylin. The data was a Kodak digital camera mounted on a light microscope. The data can be stored on a CD for later reference.

Sample analysis 85% of the samples were analyzed on a FACSCalibur flowcytometer (BDIS, San Jose, CA). Data were acquired in listmode using a threshold for nucleic acid dye fluorescence. Multiparameter data analysis was performed using Paint-A-Gate ^Pro (BDIS, San Jose, CA). Analytical criteria include those defined by forward light scatter, particle sizes defined by right angle light scatter, positive staining with PE-labeled cytokeratin monoclonal antibody and those not stained with PerCP-labeled CD45 monoclonal antibody. For each sample, the number of events present in the area typical of epithelial cells was normalized to 10 ml of blood.

  The results of isolating tumor cells added to whole blood using the assay method of the present invention are shown in FIGS. 5A and 5B. Panel 5A shows the analysis by microscopy, and panel 5B shows the analysis results obtained using flow cytometry. 6A-6C show three examples of flow cytometric analysis of a 10 ml blood sample obtained from one patient with metastatic breast cancer at three time points, with anti-leukocyte vs. anti-leukocyte analysis of flow cytometry. Includes a relative display of anti-epithelial cell antibodies. In FIG. 6, panel A, 14 events are detected, which are present in locations typical of epithelial cells. In panel 6B, 108 epithelial cells are detected, and in panel 6C, 1036 epithelial cells are detected.

  The number of events that passed the threshold set by the nucleic acid dye in the analysis of a 10 ml blood sample varied between 5,000 and 50,000 events. These events consist of cellular debris and white blood cells. In the analysis of 32 control bloods, the number of events present in the area typical of epithelial cells ranged from 0-4 / 10 ml blood (mean = 1.0, SD = 1.2).

  Eight breast cancer patients had active metastatic disease during the study period. In these patients, the number of epithelial cells in 10 ml of blood varied between 0 and 1036. Disease activity was assessed by subjective criteria, ie bone pain, dyspnea etc. and objective criteria, X-ray, bone scan, CT scan, MRI and lymph node size. Patients were classified into categories 0-4 as described in Table VI.

  The kinetics of the number of epithelial cells in the blood of 8 patients with metastatic disease is shown in FIG. The dark area in the plot indicates the range in which positive events were detected in the control. The plot also shows the period during which chemotherapy was administered. FIG. 7, Panel A shows a life-threatening disease and a patient with 200 epithelial cells / 10 ml blood when they entered the study. A high dose of adriamycine decreased the number within the normal range, but it rose again after adriamycin was discontinued. After the second course of adriamycin, the number of epithelial cells fell considerably but was still in the normal range. FIG. 7, panel B shows the course of one patient over a 43 week period. The patient was asymptomatic at the start of the study but was previously known to have bone metastases. Epithelial cells were detected above normal levels and gradually increased during the study period. A short decrease in the number of epithelial cells was found after administration of a high dose of adriamycin course. The disease activity in this patient clearly increased during this period. In FIG. 7, panels C and D, two patients are shown with less disease activity. In these patients, changes in the number of epithelial cells over time reflected changes in disease activity. In the patients shown in panels 7E and 7F, peripheral blood epithelial cell counts increased at the last time point tested, but the patient still had no symptoms.

  In the case shown in panel 7G, no epithelial cells were detected at the first time point, which was 3 years after breast cancer surgery (T2N1M0). After 4 weeks, 50 epithelial cells of 10 ml of blood were detected by flow cytometry. The patient at this point had no clinical signs of disease recurrence. Additional blood samples were analyzed to obtain morphological confirmation that the cells detected by flow cytometry had characteristics consistent with those of malignant cells.

  FIG. 8A shows two cells positively stained with cytokeratin using a large nucleus-to-cytoplasm ratio, both features consistent with tumor cells of epithelial cell origin. Four weeks after this discovery, the patient had an axillary lymph node biopsy. Cells obtained from the biopsy were found to be of malignant origin. X-rays at this point showed no sign of lung metastasis, but a CT scan performed 2 weeks later showed evidence of lung metastasis. The patient had no symptoms from lung metastases. The patient responded well to Vinorelbine as measured by loss of axillary lymph node involvement. The number of peripheral blood epithelial cells decreased to a level just above the normal range. 28 weeks after the start of treatment, the peripheral blood epithelial cell count increased, and the axillary lymph nodes increased in size as a result of the physical examination. Peripheral blood epithelial cell counts in these 8 patients with breast cancer metastatic disease reflected disease activity, response to treatment or lack thereof during the study period.

  The above test was performed using colloidal magnetic nanoparticles. Also in this example, the efficacy of large size magnetic beads for selection of low frequency tumor cells in blood was evaluated to determine if tumor cells could be selected using micron sized beads. However, as noted above, nanometer-sized magnetic particles are considered preferred for this application.

As noted above, the use of larger sized beads is disadvantageous.
These are as follows:
(I) The beads are too large to diffuse, thus requiring mixing for target cells and beads that are present at low frequency,
(Ii) The beads sink very quickly, facilitating the need for continuous mixing, and then (iii) large sized beads cluster around the cells, obscuring the analysis.

  Thus, large sized beads need to be removed from the cell surface prior to visualization or analysis. In accordance with the present invention, it has been found that the potency of cell selection with larger beads increases the bead concentration and increases the incubation time with continuous mixing to promote binding to rare target cells. In this example, 2.8 μm Dynal anti-epithelial cell beads (Dynal, NY) were used to test the efficacy of tumor cell selection from blood in a model test under optimal conditions for large beads. These beads are conjugated with a monoclonal antibody specific for epithelial tumor cells. A known number of tumor cells (cancer cell line) is added to normal blood and after selection with beads, recovery is determined. Tumor cells were labeled with a fluorescent dye to distinguish them from blood cells during detection. The manufacturer's recommended protocol was followed.

Whole blood (5 ml) was added with 15 ml polystyrene centrifuge tube followed by 20 ± 3 fluorescently labeled SKBR-3 (breast tumor cell line) cells. SKBR-3 cells were prestained with a nucleic acid staining dye (Hoechst 33342) to allow detection after selection with beads. The blood was diluted with 5 ml Dulbecco's PBS containing 5 mM EDTA and mixed with diluted blood on a rocker at 4 ° C. for 15 minutes. 100 μl of dynal anti-epithelial cell beads containing 50 × 10 ⁶ beads were added to the blood sample and incubated for 30 minutes at 4 ° C. with mixing on a rocker. Note that the number of beads used is similar to total white blood cells, ie, one bead per white cell. Magnetically labeled cells were separated by placing sample tubes in a Dynal MPC magnetic separator for 6 minutes.

  After aspirating the suspension, the collected cells were resuspended in 3 ml of Dulbecco's PBS containing 0.1% BSA. Sample tubes were returned to the Dynal MPC for 6 minutes to remove any carryover blood cells. The magnetically bound cells were resuspended in 200 μl of Dulbecco's PBS containing 0.1% BSA after aspiration of the supernatant.

  Tumor cells were detected by spotting a final sample containing selected tumor cells, non-specifically bound blood cells and excess free magnetic beads on an immunofluorescent slide. 200 μl of sample was spotted in 10 different wells to disperse the free magnetic beads. Fluorescently stained fluorescently stained tumor cells present in each well were counted using a fluorescence microscope. The results are shown in Table V:

This result shows that an average of 67% of the added cells were recovered from the blood by Dynal magnetic beads. This indicates that tumor cells present in the blood can be effectively selected with larger size magnetic beads under optimal conditions. However, in this example, only the selection of tumor cells from the blood was evaluated without performing any analysis. Furthermore, the efficiency of recovery could be determined because the cells were pre-labeled with a strong fluorescent dye. The final sample (200 μl) contained 50 × 10 ⁶ beads in addition to selected tumor cells (10-17) and non-specifically bound leukocytes. The bead size (2.8 μm) resembled certain blood cells and occupied most of the surface area on the slide. Thus, in order to obtain recovery data, the sample must be spotted in several wells to disperse the free magnetic beads sufficiently to allow detection of the recovered tumor cells.

  There are also a large number of beads on the cell surface, which eliminates the observation and staining of selected tumor cells for further analysis. In this example, tumor cells were prestained with a fluorescent nucleic acid dye and no further staining was required for detection. However, it is often desirable to identify the source tissue of magnetic bead-bound cells. Such identification is performed using labeled antibodies to detect and characterize tumor cells present in clinical samples. Thus, the beads must be removed from the cell surface and separated from the sample after target cell selection, ie, prior to analysis. This is not the case with magnetic nanoparticles. This is because their size does not interfere with cell analysis.

  In summary, this example shows that large magnetic beads can be used in the methods disclosed herein for effective isolation of circulating tumor cells.

  There are several methods available for releasing beads from the cell surface that do not significantly damage the isolated cells. One method is to replace antibodies from the cell surface by adding an excessive excess of specifically competing reagents with high affinity for the relevant antigen or antibody. This type of mechanism is used to release beads from C34 selected cells in clinical applications using a peptide (Baxter Isolex 300). The peptide competes with the CD34 antigen to bind to the antibody on the bead, releasing the antibody-bead complex from the cell. Another method uses a reversible chemical linker between the beads and the antibody.

  The chemical linker can be inserted during conjugation of the antibody to the magnetic beads. The chemical link can be cleaved under suitable conditions to release the beads from the antibody. One currently used method uses a nucleic acid linker to link the antibody to the magnetic beads. Nucleic acid linkers are polynucleotides that can be hydrolyzed, particularly using DNase enzymes. Following hydrolysis of the nucleotide bonds present in the nucleic acid linker, the beads are released from the antibody that maintains binding to the cells. The released beads can be removed from the cell suspension by magnetic separation. Cells liberated from the beads can be used for further analysis by microscopy or flow cytometry.

  This example can also isolate tumor cells from blood using large size magnetic beads, although they are used at a concentration high enough to label the cells and then released from the cells prior to analysis It shows that.

Example 4
Counting circulating epithelial cells in patients with no treatment evidence of breast cancer after surgery for willingness to treat Peripheral blood of 37 patients between 1 and 20 years after surgery was analyzed for epithelial cells by flow cytometry The existence was evaluated. Up to seven peripheral blood samples were collected from these patients over a one year period. In Table VI, each patient was listed and sorted by their TNM (tumor, lymph node and distant metastasis) at the time of surgery. Table VI also showed whether patients received treatment (either chemotherapy or hormonal treatment) during the study period. In 3 out of 6 patients with evidence of distant metastases in the past but completely relieved at the time of the study, epithelial cells were found in the blood more frequently than found in the control group. Circulating epithelial cells were found in 9 of 31 patients with no evidence of distant metastasis.

  The low number of events present in areas typical of epithelial cells by flow cytometry in these nine patients does not guarantee that these events are identified as tumor cells. The cytology obtained by placing immunomagnetically selected cells on the slides assists in assessing their identity as illustrated in FIG. FIG. 8, panel A shows two cells obtained from a patient that stained positive for cytokeratin and had no evidence of metastatic disease at the time the blood was collected. Panel 8B shows cells from a divine patient who had a metastatic disease in the past but was completely relieved. Panels 8C and 8D show that 2 cells were separated from patient 25 blood at time point 6. The cells shown in panel 8C have features consistent with malignancy, but the cells in panel 8D have the appearance of normal squamous epithelial cells.

Example 5
Circulating epithelial cell counts in patients diagnosed with breast cancer prior to surgery intervention Table VII was obtained after a similar clinical trial in which 13 controls and 30 breast cancer patients were evaluated by the assay method of the present invention. Summarize the results. In control individuals, the number of epithelial cells in 20 ml of blood is in the range of 0-5 (mean 1.5 SD = 1.8). In contrast, an average of 15.9 SD = 17.4 epithelial cells are present in a 20 ml blood sample of 14 breast cancer patients (patients classified as T _x N ₀ N ₀ ) restricted to organs. For those with lymph node involvement, 47.4 SD = 52.3, and for those with distant metastasis, 122 SD = 140. Cells between the control group with and without metastasis and between breast cancer patients were highly dominant [P, 0.001 by multiparameter analysis (Kruskal-Wallis)]. The difference between the organ-localized and distant metastasis groups was 0.009 (t test). The number of epithelial cells in patients with organ-localized breast cancer exceeded the cut-off point in 12 of 14 patients (control group = mean value in 6.9 + 3SD). Furthermore, there were no individuals in the control group that exceeded 5 events classified as epithelial cells, and only 2 out of 14 patients with organ-localized breast cancer had <7 such events.

  Flow cytometry was used to analyze positive events obtained from 20 ml blood from control individuals and women with breast cancer. The number of epithelial cells in the control blood is a statistical difference by t-test (P ≦ 0.01) and Kruskal-Wallis nonparametric analysis (P> 0.001) from each of 3 groups of breast cancer patients. The data in this table was used to establish a preliminary cutoff value for positive samples. This value is determined by averaging the number of circulating epithelial cells in a normal control (n = 13) and adding SD three times. Its average is 1.5 and its SD is 1.8. Cut-off: 1.5 + 5.4 = 6.9. There was no statistical difference between men and women.

Example 6
Monitoring CTC in patients with metastatic CAP Three patients with prostate metastatic disease were evaluated for the presence of circulating epithelial cells in their blood following chemotherapy treatment. The results are shown in FIG. The data reveal that an increase in circulating epithelial cells in the blood correlates with disease activity.

  Ten patients were selected for continuous CTC and PSA testing at intervals of 0, 1, 2, 7, 12, 17 and 25 weeks. Eight patients had hormonal refractory disease, one refused hormonal treatment, and one had hormone sensitive disease. Patient CTC and PSA levels are shown at each time point in FIG. The hormone sensitive patient had a PSA level of less than 0.1 ng / ml during the study period, and the CTC count was comparable except at the last time point when 14 tumor cells were measured (FIG. 10A). Its CTC count repeated after 1 week was 15, confirming early observation. Signs and symptoms that showed disease progression were not observed in this patient. Three patients had slow disease progression (FIGS. 10B-D). Mean CTC counts in these patients were statistically different from the control group (3.0 ± 3.0 tumor cells / 7 ml, P = 0.002, n = 25). In 6 of the 25 samples, the CTC number was 5 or more per 7 ml. Its CTC size was assessed by forward light scattering, and 77% ± 15% of these cells were larger than 10 μm in samples containing 5 or more CTCs. Six patients had disease progression during the study period. The CTC and PSA values of 4 of these patients are shown in FIGS. 10E-H. Mean CTC counts were clearly different from the control group (range 1-283, n = 22, average 45 ± 65 CTC / 7 ml) and statistically different from patients with progressive disease (P = 0.008). In 19 of the 22 samples, the CTC count was 5 or more and 51% ± 17% CTC was greater than 10 μm. In this group of patients, the increase in CTC paralleled the increase in PSA levels.

  Two patients receiving estramustine and taxane-based chemotherapy had a control group and slow disease progression (range 14-218, average 104 ± 68 CTC / 7 ml) There was a clear difference in CTC counts compared to patients. Only 32% ± 13% of CTCs in these patients had a larger size of 10 μM. Comparison of CTC sizes between the three patient groups by t-test is as follows: groups 1 and 2 (P = 0.004), groups 1 and 3 (P = 0.0001), and groups 2 and 3 (P = 0.0008). ) Showed a significant difference. CTC and PSA values and chemotherapy administration are shown in FIG. 11 for both patients. The variation in CTC was consistent with chemotherapeutic agent administration. The relative change in CTC was asserted from that of PSA, and its CTC count paralleled the PSA level in both graphs. However, the actual correlation was poor (FIG. 11A, R = 0.17 and FIG. 11B, R = 0.46).

The method of the present invention was able to quantify CTC and was used to evaluate CTC changes during HRPC progression. In vitro PC3 addition experiments showed a strong linear correlation (R ² = 0.99) and excellent recovery (74% ± 9%). The limit of detection of 0.8 ± 1.2 cells in 7.5 ml of blood was determined by analysis of blood from 22 normal male donors.

A 1 gram tumor sheds about 10 ⁶ cells into the blood. Our observation that CTCs are found in local CAP is in agreement with previous examples of evaluating localized breast cancer. Ten patients with metastatic CAP were selected to undergo serial testing for CTC load and serum PSA levels over a 6 month period. Patients were tested weekly for 3 courses and then every 5 weeks for 6 months. Four patients with either early hormone resistant prostate cancer (HRPC) (n = 3) or hormone sensitive disease (n = 1) were evaluated. Their CTC count was low (3.0 ± 3), but significantly different from the control group (P <0.002). Overall, the CTC count trend did not rise dramatically and matched the PSA pattern with the exception of patients with hormone-sensitive disease (FIG. 10A). In this case, the CTC count increased rapidly to 14 cells / 7 ml blood at week 17 and was confirmed after 1 week, suggesting that it was a pure biological event. Serum PSA levels remained undetectable. It is determined from this whether a rapid increase in CTC levels precedes the failure of biochemical PSA.

  The other four men had rapidly progressive metastatic disease or HRPC. Their CTC count was significantly higher than the early HRPC group (FIGS. 10E-H). Serum PSA and CTC values appeared to be related. However, their calculated correlations changed (FIG. 10A, R = 0.42; FIG. 10B, R = 0.67; FIG. 10C, R = 0.65 and FIG. 10D, R = 0.98). A more intrinsically different correlation (R = 0.42) occurred in the patient shown in FIG. 10E, who died of uremic coma from metastatic CAP, because of posterior disease of the large peritoneum Caused obstructive urinary tract disease. The inventors propose that metabolic abnormalities can cause a sudden and temporary decrease in CTC counts in the first week. The strongest correlation occurred in the patient shown in FIG. 10H (R = 0.98). This patient had an increased level of PSA that closely resembled a dramatic increase in CTC count. He developed a symptomatic progression but a decrease in chemotherapy.

  Two out of 10 patients received chemotherapy and had an impact on both serum PSA levels and CTC counts in a similar manner (Figure 11). These patients maintained CTC numbers in the range of 14 and 218 tumor cells / 7 ml. In both cases, the inventors observed a substantial decrease in CTC counts one week after the first Taxotere dose, which paralleled the decrease in PSA levels. Both patients showed significant increases in CTC and PSA levels despite continued dosing of Taxotere. The patient shown in FIG. 11B was then switched to taxol, and the patient shown in FIG. 11A was continued with taxotere. They both showed a decrease in CTC count between weeks 7 and 12, which was paradoxically related to either elevated or unchanged PSA levels. Eight weeks after completion of chemotherapy, increased levels of CTC and PSA were seen. These observations indicate that CTC counts can provide independent prognostic information compared to PSA levels.

  The average CTC size was smaller in people with more advanced disease. Chemotherapy changed numbers, but not CTC size, and the inventors found no change in CTC size to suggest cell depletion before, during or after chemotherapeutic agent administration.

  The inventors concluded that CTC counts are measured reproducibly in patients with HRPC. Changes in CTC levels mirrored disease progression. The pattern and rate of CTC and PSA elevation are different, suggesting that CTC provides independent prognostic information for PSA. More importantly, CTC genotype and expression characterization can guide future treatment and elucidate the mechanisms of chemosensitivity and resistance.

Example 7
Disease activity can be correlated with circulating epithelial cells in colorectal cancer patients. The analytical methods of the invention can be used to benefit the evaluation of patients with a variety of different cancer types. To illustrate, the method was also used to evaluate circulating epithelial levels in patients with colorectal cancer. There are over 130,000 new cases of colorectal cancer diagnosed each year in the United States. 30-50% of these patients will relapse and die from their disease. The rational development of new treatments is hampered by the rare availability of pre- and post-treatment tumor biopsies to demonstrate drug efficacy.

  Colorectal cancer patients with no evidence of metastasis were evaluated for the presence of circulating epithelial cells before and after surgery. The results are shown in FIG. 12 and summarized in Table XI. The data reveals that the number of circulating epithelial cells in patients with colorectal cancer is greater prior to surgical intervention.

  Table X and FIG. 13 show the data obtained when colon cancer patients with evidence of metastasis were evaluated for the presence and number of circulating epithelial cells. The results revealed that the number of epithelial cells in peripheral blood was higher in patients with metastatic disease compared to local disease after surgery. Furthermore, the results indicate that the extent of metastatic disease can be related to the number of circulating epithelial cells.

  The rational clinical development of anticancer agents is hampered by rare access to repeat tumor biopsies for in vivo pharmacodynamic evaluation. The method of the present invention overcomes this limitation by allowing assessment of drug efficacy in circulating tumor cells. Additional evaluation of the ability of the present immunomagnetic separation and automated fluorescence microscopy system to isolate, count and characterize circulating epithelial cells from peripheral blood of patients with metastatic colorectal cancer (pts.) Preliminary studies were conducted. Twenty patients with measurable metastatic disease were used. Five ml of peripheral blood was obtained at the start of treatment and at the time of disease reassessment (6-10 week interval). In addition, fresh tumors were obtained in 4 patients for comparison of circulating and in situ cancer cells by flow cytometry and gene sequencing. Patient characteristics were: 7M / 13F, median age 64 (range 41-80), median time 2.7 m (range 0.6-25 m) with metastatic disease. Eleven patients received prior chemotherapy for metastatic disease. Sites of metastatic disease included liver (13 patients), lungs (8 patients), peritoneum (5 patients), small intestine and anterior abdominal wall (1 patient each). The median diameter of the largest metastatic lesion was 5 cm (range 1.5-12 cm). Circulating epithelial cells were purified from whole blood after labeling with anti-epithelial cell adhesion molecule (EpCAM) conjugated to ferrofluid. The median number of recovered epithelial cells was 7 / 7.5 ml peripheral blood (range 3 to 150) measured by flow cytometry labeled with anti-cytokeratin. The test results are shown in Table IXB.

  In addition, further phenotyping by flow cytometry for epidermal growth factor receptor and thymidylate synthase expression to assess the suitability of this technique for in vivo pharmacodynamic evaluation is also provided by the present invention. It can be done according to the method. This study showed the feasibility of isolating circulating tumor cells from the blood of patients with metastatic colorectal cancer. In addition, the present invention includes a method of assessing changes in circulating tumor cells relative to tumor cells that are in situ in a tumor population. Such changes may include, for example, the acquisition or loss of tumor constitution related molecules. Also, changes in genotype or expression type can be examined.

  The above examples show a highly significant difference in the number of circulating epithelial cells between healthy individuals and patients with breast, prostate and colon cancer. In addition, significant differences in the number of circulating epithelial cells were found between patients who did not detectably spread, patients who had spread to local lymph nodes, and patients with distant metastases (Racila et al., (1998), supra). . In addition, the number of epithelial cells in the patient's blood after surgical removal of early breast cancer was monitored for one year. In some of these patients, the remaining disease was detected. In patients with metastatic disease, changes in peripheral blood tumor cell counts were associated with tumor burden and response to treatment. These test results demonstrate the potential of the cell-based assay method of the present invention as an objective non-invasive tool for detecting the presence of malignant diseases and measuring disease activity. Cell morphology and immunophenotype reveal the malignant nature of isolated cells.

Example 8
Identification of the tissue source of isolated epithelial cells All of the above studies in patients have excess epithelium circulating in patients with cancer compared to normal individuals or patients without cancer disease, including benign tumors. Reveal that cells are present. However, it is essential that these excessive circulating epithelial cells are in fact found to be tumor cells. This is accomplished by performing experiments in which immunomagnetically purified epithelial cells from patients with or without cancer are cytospun onto glass slides and treated with anti-mucin. In addition, normal epithelial cells obtained from foreskin and blood from normal individuals who used both as controls were cytospun. It is important that the slide be encoded and evaluated “blind”, that the observer trains in pathology, and includes normal epithelial cells. As can be seen in FIG. 8, there is a significant difference between isolated tumor cells-versus normal epithelial cells. Normal epithelial cells have a low nucleus-to-cell ratio, ie there is an abundant cytoplasm and relatively small nuclei. The nucleus shows a smooth distribution of chromatin. Cells are not stained with anti-mucin. In contrast, cells from two patients with breast cancer have a very large nucleus and a small periphery of the cytoplasm. In addition, the chromatin is confused as shown by the nuclear patch, and the cells are strongly stained with anti-mucin. The same is observed in cells from two patients with prostate cancer. Coded slides from patients with and without cancer were shown by physicians trained in pathology (a total of 21 slides). Physicians trained in pathology correctly identified blood from controls because they had no cancer cells when showing slides twice and showed no internal observation errors. In the case of 2 patients with prostate cancer, tumor cells were not seen in the study. One slide was re-evaluated and tumor cells were observed. The cause of this discrepancy appears to be the amount of time spent scanning the cell smear. In summary, cytomorphology and immunophenotype indicate that excess epithelial cells present in the blood of patients with cancer are in fact cancer cells.

From the above experiments, the method disclosed herein enables the detection of cancer cells in the blood of patients with early tumors. In fact, in 25 of the 27 patients clinically determined to have organ-localized disease (early cancer), the inventors detected the presence of cancer cells in the blood. This means that the assay method will detect cancer cells very early in those solid tumors which are usually detected late (10 ⁹ -10 ¹⁰ tumor cells). In addition, the test will allow for the detection of breast, prostate and colon cancer by conventional means early, possibly before detection of early tumors. The organ source of tumor cells in the blood for the prostate should be stained with anti-prostate specific cell membrane antigen (PMSA), anti-PSA (prostate specific antigen) or other antibodies specific for the prostate of a male subject Can be established. In breast cancer in female patients, staining with anti-mammoglobin anti-progesterone receptor, anti-estrogen receptor and anti-milk fat globulin antigens I and II will indicate a tumor of breast origin.

  Our test will detect cancer cells from other organs such as pancreas, esophagus, colon, stomach, lung, ovary, kidney and the like. The following table shows examples where excess epithelial cells were observed in some patients with adenocarcinoma other than breast and prostate.

  Each carcinoma listed in the table expresses a tissue-specific antigen that, using its corresponding antibody, can determine the organ source of circulating tumor cells.

  A diagram showing the process of providing early tumor cells progresses to metastatic cancer in FIG. The blood test of the present invention can also be used to detect cancer cells in patients who have been successfully treated for cancer in the past and are currently in full remission for a long time. In fact, circulating epithelial cells, ie, dormant tumor cells, have been detected in patients who have been previously treated for more than 5 years and are likely to have clinical release of the tumor. This explains why the patient can have many years of recurrence, even after 10 years after a clearly successful treatment. In fact, the accumulation of evidence suggests that recurrence of breast cancer occurs at a slow and steady rate approximately 10-12 years after mastectomy.

Example 9
Detection of tumor cells in the blood in patients with high PSA levels and negative biopsies As demonstrated by the previous examples, the present invention can be used to diagnose cancer in patients who are currently asymptomatic. Bring. To illustrate this point, patients with a 2-year history of high PSA levels (> 12 μl / ml) had a prostate needle biopsy performed 2 weeks prior to the analysis described below. The biopsy did not reveal the presence of malignancy. It should also be noted that the previous biopsy performed 18 months ago was negative.

  Prior to obtaining a 20 ml blood sample, the patient was given a digital rectal exam intended to increase the incidence of tumor cells in the blood and a gentle massage of his enlarged prostate. Blood samples were enriched using the method of the present invention. Enriched fractions were examined by microscopy using Wrights-Giemsa staining. Morphological examination of the isolated cells revealed their malignant characteristics. Obviously, this patient had cancer. The diagnosis of prostate cancer is likely to give the observed high PSA levels. The source of the cells can be determined using appropriate reagents as described herein. The results presented in this example demonstrate that the method of the present invention can be used to detect sources that are no longer discovered.

  The idea of using a local massage to encourage the flow of tumor cells into the blood as a means of enhancing the sensitivity of blood tests is a concept with considerable advantages. Following isolation, cells released into the circulation by this approach can be used for a variety of different purposes. In the case of cells isolated with ferrofluid, the isolated cells can be easily cultured and / or cloned. The resulting cell lines can be used to evaluate various malignant cell properties such as chemosensitivity and growth factor dependence.

Example 10
Monitoring biochemical changes in isolated circulating tumor cells Selective, which can lead to dominant phenotypic changes in tumor cells over time as a result of genetic instability of tumor cells in cancer development Create new clones with growth advantages. One such example is amplification of the HER-2 (c-erbB2) proto-oncogene, resulting in overexpression of the encoded epidermal growth factor transmembrane receptor. The purpose of this study was to determine whether HER-2 receptor can be quantified with respect to circulating tumor cells (CTC) in the blood of patients with advanced breast cancer, and the pattern of HER-2 expression changed during treatment Is to decide what to do. This information will allow clinicians to predict the outcome of expensive new targeted treatments. Although HER-2 is exemplified herein, it is highly desirable that additional tumor constitution related molecules on or in tumor cells be identified and evaluated in this manner. Suitable tumor constitution related molecules that can be evaluated following isolation of circulating tumor cells are listed in Table XI. Typical approaches for detecting changes in tumor constitution related molecules such as nucleic acids or polypeptides / proteins associated with malignancy include the following:
a) comparing the sequence of the predicted nucleic acid in the sample with the corresponding wild-type nucleic acid sequence to determine if the sample from the patient contains a mutation; or b) from a patient with a tumor constitutional associated molecular polypeptide And if present, determine whether the polypeptide is sufficiently long and / or mutated and / or expressed at normal levels; or c) Using DNA restriction mapping, a restriction pattern purified when a restriction enzyme cleaves a sample of a tumor predisposition-associated molecular nucleic acid from a patient is referred to as a restriction pattern obtained from a cognate normal gene or a known mutation thereof. Or d) using a specific binding member (either a normal sequence or a known mutant sequence) capable of binding to a tumor constitution associated molecule nucleic acid sequence, the specific binding member A nucleic acid sequence capable of hybridizing to the sequence of the substance, wherein the substance comprises an antibody having specificity for the native or mutated nucleic acid sequence or the polypeptide encoded thereby, and the specific binding member is the binding of the specific binding member E) Screen for normal or mutated genes in samples from patients using PCR that is detectably labeled for binding to the partner; or e) PCR that includes one or more primers based on the normal or mutated gene sequence.

  Changes in protein molecules, such as those resulting from deletions or point mutations in the encoded nucleic acid, can be assessed using conventional methods well known to those skilled in the art. Such methods include gel electrophoresis, western blot, HPLC and FPLC. In addition, changes in nucleic acid molecules associated with malignancy can be assessed using conventional methods.

  It is also possible to evaluate changes in hydrocarbon sites present on membrane glycoproteins. Protocols for analyzing sugar conjugates and sugars thereon are provided in Ausubel et al., Supra, Chapter 17.

  In the method of the present invention, surgically resected early tumor tissue is evaluated for the expression of a limited number of genes and / or proteins. Using breast cancer as an example, such tumor constitution related molecules may include HER-2, estrogen and progesterone. The problem is cited as disease progression because changes in the phenotype of the tumor cells occur after the original diagnosis and resistance to treatment is inferred after treatment failure. The assumption of the presence of target tumor constitution related molecules for CTC before and during treatment constitutes a real-time “whole body” biopsy of the tumor. These goals were used to develop counting CTCs and quantifying and characterizing the expression of tumor constitution related molecules present on or in tumor cells. In this example, we describe changes in the expression of HER-2 on CTCs from the blood of patients with metastatic breast cancer.

  The following materials and methods are provided to facilitate implementation of Example 10.

The patient population study was conducted at the Lombardi Cancer Center at Georgetown University. Patients in this study were selected from 24 women in a larger group of Phase III or metastatic breast cancer enrolled in a preliminary study to monitor CTC variation during treatment (Walker et al. Proc. Am Soc. Clin. Onc. (2001) 19-54b). Nineteen patients whose early tissue block could be obtained were included in this study. All patients have a measurable disease and are a newly diagnosed stage III patient who has started new adjuvant chemotherapy or a document that initiates a new endocrine, chemical or experimental treatment Who had advanced metastatic breast cancer. The choice of treatment was left to the judgment of the treating physician. Neither the treating physician nor the patient was notified of the CTC assay results. As a control, blood samples were collected from 22 disease-free women over 21 years of age. The protocol was approved by IRB. Informed consent or release was obtained from patients and normal control subjects, respectively.

Clinical Evaluation Two pre-registration evaluations were performed where possible, the first was study entry within 4 weeks, and the second (baseline) was just before the start of treatment. A current medical history was obtained and then a medical examination was performed by a clinical oncologist. Hematological analysis included CBC with difference and platelet count; biochemistry including urinalysis, BUN, GOT, GPT, LDH, creatinine, alkaline phosphatase and total / direct bilirubin. Tumor assessment was based on physical measurements (Caliper or ruler) and / or imaging studies (CT scan, MRI, bone scan, etc.). Evaluation of response to treatment is defined using the Union International Contra Cancer (UICC) criteria (Monfardini et al., UICC- Manual of Adult and Pediatric Medical Oncology, Berlin, Germany, Springer 1987, p22-38), and CTC / CTC HER- 2 Done without knowledge about the results. The timing of blood collection and evaluation of clinical status depended on the individual treatment protocol. Blood samples were collected in 10 ml ACD Vacutainer tubes (Becton Dickinson, NJ) all maintained at room temperature and run overnight on Imunicon. Samples were processed within 24 hours after collection.

HER-2 stained tissue blocks Tissue blocks from early patient tumors were collected and slides were prepared from paraffin-embedded tissue sections. According to the manufacturer's instructions The slides were evaluated for HER-2 expression using ^{HercepTest R (DAKO, Carpinteria, CA} ). Positive and negative slides were evaluated by one pathologist and scored according to manufacturer's guidelines using a scale of 0 to 3+.

Sample Preparation 5 ml of blood was transferred to a disposable test tube (Fisher Scientific, USA) with an internal diameter of 17 mm and centrifuged at 800 g for 10 minutes with brake off. Phosphate buffered saline (PBS) containing bovine serum albumin was added to bring it to a volume of 10 ml and the sample was mixed by inversion. As noted above, monoclonal antibodies (Mabs) specific for epithelial cell adhesion molecules (EpCAM) are broadly reactive with tissues of epithelial cell origin. Mab VU-1D9 recognized EpCAM and coupled to magnetic nanoparticles (ferrofluid, Immunicon, Huntingdon Valley, PA). Coupling Desthiobiotin to EpCAM-labeled magnetic nanoparticles to increase the “magnetic load” of EpCAM + cells and to reduce variations in capture efficiency due to differences in EpCAM density on the cell surface The CA-EpCAM described in the previous examples was formed. CA-EpCAM ferrofluid and buffer containing streptavidin were then added to the sample to achieve this increase in magnetic labeling of the cells. Desthiobiotin on the CA-EpCAM ferrofluid was subsequently replaced by biotin contained in the permeabilization buffer described below, thereby transforming the crosslinks between the CA-EpCAM ferrofluids. Immediately, the sample was placed in a magnetic separator composed of 4 opposing magnets for 10 minutes (QMS 17, Immunicon, Huntingdon Valley, PA). After 10 minutes, the test tube was removed from the separator, inverted 5 times, and returned to the magnetic separator for an additional 10 minutes. This process was repeated once more and the test tube was returned to the separator for 20 minutes. After separation, the supernatant was aspirated and discarded. The test tube was removed from the magnetic separator and the fraction collected on the vessel wall was resuspended in 3 ml of BSA-containing PBS. The suspension was placed in a magnetic separator for 10 minutes and the supernatant was aspirated and discarded. Cells were resuspended in 200 μl of biotin-containing permeation buffer (Immuniperm, Immunicon Corp.) supplemented with Mab fluorochrome conjugate at saturation conditions. The Mab is anti-cytokeratin MabC11 conjugated with phycoerythrin (PE) (Immunicon Corp.), which recognizes keratins 4, 6, 8, 10, 13, and 18 and anti-labeled with Peridinin chlorophyll protein (PerCP). It consisted of CD45 (Hle-1, BDIS, San Jose, CA) and cyanine 3 (CY-5) labeled anti-HER-2. MAb anti-HER-2, designated HER-81, recognizes an epitope on the extracellular domain of HER-2 and does not cross the block with trastuzumab or its murine parent 4D5. It is a mouse IgG1k with a Kd of 10 ⁻¹⁰ M against BT474 breast cancer cells. After incubating the cells with the Mab for 15 minutes, 2 ml of cell buffer (PBS, 1% BSA, 50 mM EDTA) was added and the cell suspension was magnetically separated for 10 minutes. After discarding the unseparated suspension, the collected cells were resuspended in 0.5 ml PBS and the nucleic acid dye used in the Procount system was added (Procount, BDIS, San Jose CA). In addition, 10,000 fluorescent count beads were added to the suspension to confirm the amount of sample analyzed (Flow-Set Fluorospheres, Coulter, Miami, FLA).

Cell Lines Prostate tumor cell line PC-3 and breast cancer line SKBR-3 cells were cultured in flasks containing 10 ml RPMI-1640 supplemented with 10% FCS. Cells were harvested from the flask after trypsinization, washed and resuspended to obtain the desired cell concentration. For quantitative evaluation of HER-2 against both cell lines, 20,000 cells were stained with MabHER-81 conjugated to PE (HER-2 PE). For HER-2 expression level calibration, 100 μl of cell suspension containing about 3,000 cells was added to 5 ml of blood.

Sample Analysis Samples were analyzed on a FACS Calibur flow cytometer equipped with a 488 nm argon ion laser and a 635 nm laser diode (BDIS, San Jose, Calif.). Data collection was performed at CellQuest (BDIS, San Jose, Calif.) Using thresholds for nucleic acid dye fluorescence. After analyzing 8000 beads or 80% sample, the acquisition was stopped. Multiparameter data analysis was performed on list mode data (Paint-A-Gate ^Pro BDIS, San Jose, CA). Analytical criteria included size defined by forward light scatter, particle size defined by right angle light scatter, positive staining with PE-labeled anti-cytokeratin MAb and those not stained with PerCP-labeled anti-CD45 Mab. For each sample, the number of events present in the region typical of epithelial cells was multiplied by a correction factor of 1.25 to account for the amount of sample not analyzed by flow cytometry. The flow cytometer was calibrated to assess the density of HER-2 on the cells using phycoerythrin (PE) labeled beads containing a known number of fluorophores (QuantiBRITE PE, BDIS, San Jose, Calif.). The density of HER-2 on the breast tumor cell line SKBR-3 and the prostate tumor cell line PC-3 was then measured by measuring the fluorescence intensity of cells treated with PE-anti-HER-2 under saturation conditions. evaluated. The average number of HER-2 receptors on SKBR-3 and PC-3 cells was 850,000 and 9,500, respectively.

CTC counting In order to obtain the sensitivity required for CTC counting, a combination of techniques was required. First, samples enriched in EpCAM + cells in blood were prepared by mixing cells with colloidal paramagnetic particles coated with Epbs specific MAbs and subsequent magnetic separation. This enrichment of the immunomagnetic sample was performed to reduce both the sample volume and the number of background hematopoietic cells. The remaining cells are then labeled with PE-anti-cytokeratin to identify epithelial cells, labeled with PerCP-anti-CD45 to identify leukocytes, labeled with Cy5-anti-HER-2 and labeled with HER-2. Expression was determined and then nucleic acid dyes were added to rule out “events” with no remaining red blood cells, platelets and other nuclei. Magnetic separation (ie, washing) was used throughout sample preparation to remove excess labeled reagent, reduce carryover of non-target cells, and allow cell resuspension in the desired volume. The sample was analyzed by flow cytometry with each event characterized by two light scattering and four fluorescence parameters. FIG. 15 (Panels AD) is an example of flow cytometric analysis of a 5 ml blood sample obtained from a patient with metastatic breast cancer. Panel 15A shows a relative display of PerCP-anti-CD45-vs-PE-anti-cytokeratin; panel 15B shows PE-anti-cytokeratin-vs-nucleic acid dyes; Shows right-angle scatter, panel 15D shows Cy5-anti-HER-2-vs.-PE-anti-cytokeratin. The location of the beads and white blood cells are shown in the panel and are drawn in black. The gates depicted in panels 15A, 15B and 15C show regions typical for CTC (CD45 ⁻ , cytokeratin ⁺ ,> 4 μm and nucleic acid ⁺ ). To qualify as a CTC (highlighted dark little square), the event had to be in all three areas. All other events consist of debris and are drawn in gray. CTCs were considered HER-2 ⁺ if they exceeded the white blood cell background staining as indicated by the dotted line in panel 15D. All samples in this test were analyzed using the same gate shown in the figure. In 5 ml blood from 22 controls, 1.5 ± 2.1 events per subject were detected in the area classified as CTC. None of these events were related to HER-2 expression. In contrast, 5-214 CTC / 5 ml were detected in 10 blood samples out of 19 from patients with measurable disease that started the new treatment or started a new system.

Expression of HER-2 on CTC To measure the expression level of HER-2 on CTC, SKBR-3 (about 850,000 HER-2 receptors) and PC-3 (about 9 , 500 HER-2 receptors) cells were added and then processed for CTC analysis. Leukocytes, PC-3 and SKBR-3 cells were identified based on a unique profile of cytokeratin and CD45 expression. The fluorescence intensity of Cy5-anti-HER-2 staining of each cell population is shown in FIG. 16A. The expression level of HER-2 on CTC was subdivided into four categories;
(1) No expression, [less than 5,000 receptors (−)] typical for leukocytes, (2) Low expression [5,000-50,000 receptors] (+) typical for PC-3 (3) Moderate expression [50,000-500,000 receptors] (++) and (4) High expression [> 500,000 receptors (++++)] typical for SKRB-3 . The expression of cytokeratin and HER-2 on CTCs from 3 breast cancer patients is shown in FIGS. 16B, 16C and 16D. Most CTCs from patients shown in panel 16B had low levels of expressed HER-2, but a small number were completely devoid of HER-2. All levels of HER-2 expression were found on the patient's CTC as described in panel 16C. The CTC in the patient shown in panel 16D appeared to be divided into a CTC expressing no or low levels of HER-2 and a CTC expressing high levels of HER-2. In all cases, CTCs that expressed high levels of HER-2 expressed relatively low levels of cytokeratin. Table XI shows the treatment format, response to therapy, HER-2 expression on primary tissue, months between evaluation of tissue biopsy and CTC, baseline and post-treatment CTCs for CTCs from 19 breast cancer patients And HER-2 expression. Early tumor tissue blocks obtained at diagnosis were assayed for HER-2 expression. The time between diagnosis (tumor biopsy) and CTC determination varied greatly (Table XI). Tissue block of 7 out of 19 patients was positive for HER-2 (2+ or 3+) by Herceptest staining. In 6/7 patients, CTC was detected and in all 6 the CTC expressed HER-2 (Table XII). In one patient, CTC expressed HER-2 but no tissue sections. The percentage of CTC that expressed HER-2 and the density of HER-2 on the surface of the CTC varied considerably.

Ｐｔ＃＝患者番号、応答カテゴリー：Ｐ＝進行、Ｒ＝完了または部分的応答、Ｓ＝安定、ＲＸ＝処置：Ｄｏ＝ドキソルビシン、Ｃｙ＝シクロフォスファミド、Ｆｌｕ＝フルオキシメステロン（fluoxymesterone）、Ｇｏ＝酢酸ゴセレリン、Ａｎ＝アナストロゾール（anastrozole）、Ｅｘｅ＝エグセメスタン（exemestane）、Ｍｅｇ＝酢酸メゲステロール（megestrol）、Ｔａｍ＝タモキシフェン、Ｃａｐ＝カペシタビン（capecitabine）、Ｔａ＝パクリタキセル（Paclitaxel）、Ｔｒ＝トラスツズマ（trastuzumab）；期間（月） 組織／ＣＴＣｓ＝組織生検およびＣＴＣ評価の間の期間（月）。ＣＴＣ：−＝＜５個のＣＴＣ／５ｍｌ血液；＋＝５−１０個のＣＴＣ／５ｍｌ血液；＋＋＝１０−１００個のＣＴＣ／５ｍｌ血液；＋＋＋＝１００−１０００個のＣＴＣ／５ｍｌ血液。ＣＴＣ上のＨｅｒ−２：−＝＜２５％のＣＴＣ発現ＨＥＲ−２；＋＝２５−５０％のＣＴＣ発現ＨＥＲ−２；＋＋＝５０−７５％のＣＴＣ発現ＨＥＲ−２；＋＋＋＝７５−１００％のＣＴＣ発現ＨＥＲ−２；ｎａ＝ＣＴＣが検出されないので適用可能できない。概観の目的で、発明者らは、ＨＥＲ−２発現をＨＥＲ−２を発現しているＣＴＣのパーセンテージとして分類した。後記の図において、４つの異なる密度のＣＴＣの実数を異なる時点にて示す。

Pt # = patient number, response category: P = progression, R = complete or partial response, S = stable, RX = treatment: Do = doxorubicin, Cy = cyclophosphamide, Flu = fluoxymesterone, Go = goserelin acetate, An = anastrozole, Exe = exemestane, Meg = megestrol acetate, Tam = tamoxifen, Cap = capecitabine, Ta = paclitaxel, Tr = Trastuzumab; period (months) Tissue / CTCs = period (months) between tissue biopsy and CTC assessment. CTC:-= <5 CTC / 5 ml blood; + = 5-10 CTC / 5 ml blood; ++ = 10-100 CTC / 5 ml blood; +++ = 100-1000 CTC / 5 ml blood. Her-2 on CTC:-= <25% CTC-expressing HER-2; + = 25-50% CTC-expressing HER-2; ++ = 50-75% CTC-expressing HER-2; +++ = 75-100 % CTC expression HER-2; na = CTC is not detected and is not applicable. For overview purposes, we classified HER-2 expression as a percentage of CTCs expressing HER-2. In the figure below, the real numbers of four different density CTCs are shown at different times.

CTC count and expression of HER-2 on CTCs during treatment Eight patients progressed, 4 had stable disease, and 7 responded to their treatment (Table XI). In 3 patients, HER-2 expression was not detected on CTC at the start of treatment. The disease progressed to all three patients during the monitored period. In subsequent measurements, CTCs increased and a percentage of CTCs expressed HER-2 (Table XI). FIG. 17 shows the number of CTCs detected in these three patients before and during treatment. The arrow indicates the time of treatment. The number of CTCs expressing HER-2 deleted (0) or low (+), moderate (++) or high (++++) is shown in the bar.

  In 3 patients with disease progression, HER-2

Were present on baseline CTCs and the percentage of CTCs that expressed HER-2 did not show a clear change (Figure 18). CTC gradually increased over the course of treatment in the patients shown in FIGS. 18A and 18B. The CTC in the patient's blood shown in panel 18C was essentially low and did not increase during the course of treatment. The number of CTCs before, during and after treatment of the other 3 patients with stable disease being followed is shown in FIG. The number of CTCs in the patient's blood shown in FIG. 19A increased, while the CTCs in the other two patients decreased but could still be detected after treatment. Of note, the patient described in FIG. 19C (patient 21) was treated with trastuzuma and paclitaxel. 98% CTCs expressed low to moderate HER-2 HER-2 prior to treatment. The number of CTCs decreased from 214 to 79 after 4 weeks of treatment and only 14% expressed HER-2. The number of CTCs continued to decrease over the course of treatment (23, 13 and 11 CTCs), and the percentage of CTCs that expressed HER-2 increased to 78, 100 and 54%, respectively. The patient's clinical status (stable disease) was based on CT and bone scan performed after the course of treatment, but a clear reduction in CTC was observed.

Discussion Recent approval of HER-2 overexpression for the treatment of women with breast cancer to Torasutsuzuma (Herceptin ^R) is, Baselga et al. Proc plus important administration to available treatments for patients with this disease. Am Soc. Clin. Onc. (1995) 14: 103a; Pegram et al. J. Clin. Oncol. (1998) 16: 2659-71; Cobleigh et al. J. Clin. Oncol. (1999) 17: 2639). The development and use of such targeted therapies requires "real time", accurate, sensitive, specific and reliable in vitro diagnostic assay methods. The assay should be able to detect not only the subpopulation of patients that would benefit from a given treatment, but also the subpopulation of patients who developed resistance to the treatment. Candidates for trastuzuma treatment are currently positive diagnosis either by immunohistochemistry (positive HER-2 staining of tumors) or evidence showing HER-2 gene amplification as determined by fluorescence hybridization (FISH) Need. Apart from the debate over the accuracy and sensitivity of both approaches, both approaches (Lebeau et al., J. Clin. Oncol. (2001) 19: 354-363; Kakar et al. Molecular Diagnosis (2000) 5: 199-207) No change in tumor phenotype is detected from the time of initial diagnosis to the time of detection of recurrence of the disease. Changes in tumor genotypes and expression types that occur during the clinical course of the disease are documented (Vogelstein et al. N. Engl. J. Med. (1988) 319: 525-532; Pihan et al. Cancer Res. (2001) 61: 2212-2219), to be determined before initiating expensive targeted therapy. Because most (> 75%) breast cancer metastases are intrinsic (bone, liver, lung, etc.), the morbidity and cost of obtaining routine biopsies is prohibitively expensive. In this example, the inventors have shown that epithelial cells isolated from blood can be used to quantitatively analyze the presence of tumor constitution related molecules. These increased cell numbers in patients with cancer can be used to assess tumor progression. A prior study by this inventor revealed that the CTC measured at several time points during the day did not change, but a substantial increase was found over a longer period of time as the disease progressed. In this study, CTCs were detected in 5 ml of blood from 10 out of 19 patients with stage III and IV breast cancer. To increase the frequency of patients in which CTC is detected, more blood and sample preparation procedures can be used to increase the sensitivity of the assay. The observation that CTCs from patients with tumors that overexpress HER-2 were HER-2 ⁺ supports the logical basis of the assay. More important was the fact that in 3 patients we observed a phenotypic conversion from HER-2- ⁻ to HER-2 ⁺ CTC. HER-2 overexpression was not detected on CTCs prior to initiation of treatment in the new system, but was detected on CTCs that received a concomitant substantial increase in the number of treatments. Conversion to HER-2 ⁺ CTC may indicate a more aggressive conversion to the phenotype. The observation that the expression of HER-2 is related to the expression of cytokeratin, a cytoskeletal protein associated with cell differentiation, supports the hypothesis (Schaafsma et al. In Rosen, PP, Fechner, RE, Pathology Annual vol. 29 (1994) pp. 21-62). Thus, the ability to detect changes in HER-2 expression is clinically important with respect to the selection of HER-2 targeted for therapy and chemotherapy.

  In summary, the inventors have shown that the clinical status of patients with breast cancer can be assessed by changes in CTC levels, and antigenic targets on these cells are quantitatively assessed. This information may be beneficial in “Tailor” treatment attempts for individual patient disease. Although HER-2 is exemplified herein, changes in many other tumor constitution related molecules and markers can occur and tumor cells can become more malignant. Molecules that show malignant related changes include but are not limited to mdr, thymidylate synthase, FSFR, p53, ras oncogene, CD146, src, MUC1, uPA, PAI-1, ACT and others Many are included. Chromosomal translocations, point mutations in key cell signaling molecules, and surface carbohydrate changes associated with cancer have all been described previously. Table XII provides a list of biological molecules that can be evaluated by the clinician, provides a more accurate diagnosis of the patient's disease, and more importantly, formulates an appropriate therapeutic regimen for treatment. For example, pS2 / pNR-2 + breast cancer indicates that the patient will respond to endocrine treatment. Additional tumor constitutions that change frequently during malignant progression and can be analyzed in isolated circulating tumor cells are listed below. This list is merely illustrative and is not limited to the molecules described.

  The methods are available to those skilled in the art to analyze changes in expression levels, metabolic functions and / or genetic changes in tumor constitution related molecules listed in XII. Changes in protein expression level can be evaluated by flow cytometry, laser scanning cytometry, immunocytochemistry, Celltrax and the like. Genetic changes such as point mutations can be assessed using restriction enzyme digestion, FISH, PCR or Western hybridization. Altered glycoprotein and glycolipid glycosylation is a common feature in cancer, possibly allowing cell-cell interactions in favor of metastasis or allowing tumor cells to avoid immune-surveillance Doing so may affect cancer cell behavior. Changes in glycosylation of human cancer can be assessed using immunohistochemical techniques, mass spectrometry and column chromatography. A schematic protocol for carrying out the method of the present invention is provided in FIG.

Example 11
Test Kits for Analyzing Various Embodiments of Cancer Also contemplated for use in the present invention are test kits that contain reagents used to perform the assays of the present invention. Such kits are designed for special applications. Reagents can be assembled to facilitate patient screening for circulating rare cells, including but not limited to tumor cells. In this embodiment, the kit includes colloidal magnetic particles comprising a magnetic core material, a protein-based coating material, and a biologically specific ligand that specifically binds to a specific determinant present on the cancer cell to be isolated. . The kit also includes at least one additional biological having affinity for a second unique determinant on the cancer cell to be isolated that is different from the determinant recognized by the biologically specific ligand. Contains determinants specific for. The kit also includes cell-specific dyes to exclude cells without nuclei and other non-target sample components from the analysis. Exemplary kits also include reagents for detecting at least one tumor constitution associated molecule. Also provided in the kit is the Cell Spotter or Celltrax cartridge described in Example 2.

  A typical kit according to the present invention can comprise an anti-EpCAM and a pair of monoclonal antibodies coupled directly or indirectly to magnetic nanoparticles, the first antibody recognizing a cancer-specific determinant. The second antibody has an affinity for non-tumor cell determinants, such as pan-leukocyte antigens. Also provided in the kit is a reagent for detecting at least one tumor constitution associated molecule. The kit also includes a nucleic acid dye to exclude cells without nuclei from analysis. The kits of the invention can optionally include biological buffers, permeabilization buffers, protocols, separation containers, analysis chambers, positive cells or appropriate beads and information sheets.

  The kits can also be manufactured to facilitate diagnosis and characterization of the specific cancer cells detected that are represented in the circulation. In this embodiment, the kit includes all of the above listed items, and preferably further includes a panel of cancer specific monoclonal antibodies. Using breast cancer as an example, diagnostic kits include anti-MUC-1, anti-estrogen and anti-progesterone receptor antibodies, anti-CA27.29, anti-CA15.3, anti-cathepsin receptor D, anti-p53, anti-urokinase type plasminogen. An activator, anti-epidermal growth factor, anti-epidermal growth factor receptor, anti-BRCA1, anti-BRCA2, anti-prostate specific antigen, anti-plasminogen activator inhibitor, anti-Her2-neu antibody or a subset of the above may be included.

  Kits are also provided to monitor disease that recurs following tumor eradication and / or remaining cells. In this embodiment, the type of cancer will have already been diagnosed. Thus, the kit includes all of the reagents utilized using a biological sample for cancer, and further includes additional antibodies specific for the type of cancer previously diagnosed in the patient. Again, using breast cancer as an example, such a kit can contain anti-MUC-1. Alternatively, the kit can contain anti-Her2-neu.

  The kit of the present invention can screen, diagnose or monitor a variety of different cancer types. For example, if the kit is used to detect prostate cancer, the antibody or complementary nucleic acid included in the kit will be specific for a target molecule present in prostate tissue. Suitable antibodies or markers for this purpose include anti-prostate specific antigens, free PSA, prostatic acid phosphatase, creatine kinase, thymosin b-15, p53, HPC1-based prostate gene and prostate specific membrane antigen. If the patient is screened for the presence of colon cancer, an antibody specific for carcinoembryonic antigen (CEA) can be included in the kit. Kits utilized for screening patients with bladder cancer can include antibodies against nuclear matrix proteins (NMP22), Bard bladder tumor antigen (BTA) or fibrin lowering product (FDP) antibodies. Markers and tumor constitution related molecules are known for a number of different cancer types.

  Cells isolated using the kit of the invention can be further tested for, but not limited to, chromatography, RNA associated with the organ of origin, surface and intracellular proteins, particularly those associated with malignancy. Based on existing information about such molecules, it may be possible to determine from the expression of isolated cells, the ability of that tumor cell through analysis of circulating cells.

It is an object of the present invention to provide kits for any cancer for which specific markers are known. A list of tumor constitution related molecules and their usefulness and / or indications are as follows:
I. Indication of Tumor Origin Muc-1--Breast PSA and PSMA--Prostate CEA--Colon CYPRA 21-1--Lung CA 125--Ovary Cytokeratin--See List Anti-HI67
II Cell Cycle Nucleic Acid Dye Cyclins A, C and E
p27
III cell viability / apoptosis bax
Bcl-2
Caspase-7
Caspase 8
Caspase 9
Fas (CD95)
Cytochrome C
Amexin V,
Antimetalloproteinase IV drug sensitivity Estrogen, progesterone and androgen receptor HER-2 / neu
V. Drug resistance
P-glycoprotein (MDR)
t-glutamylcysteine synthetase taxol-resistance-related gene-1-5
Cis-diamine dichloroplatinum II resistance gene Thymidylate synthase Protein kinase C
Telomerase

VI. Staging Lewis A
C
BRCA-1 BRCA-2
CA15.3 (Muc-1), CA 27.29, CA 19.9
LASA
p53
Cathepsin D
ras oncogene

  The following table provides different cytokeratin markers that can be used to evaluate the tissue source of cells isolated using the methods of the invention.

  The following shows why the implementation of the method of the invention is facilitated by a kit for use in the detection of circulating breast tumor cells.

As noted above, the kit begins with reagents, devices and methods for enriching tumor cells from whole blood. An exemplary kit for detecting breast cancer cells includes assessing six factors or indications. The analysis platform requires that the reporter molecule be positioned such that it is distinguished by appropriate excitation and emission filters DAPi, CY2, CY3, CY3.5, CY5 and CY5.5. The analytical platform in this example uses a mercury microscope equipped with a mercury arc lamp and a suitable filter set for evaluating the wavelength of the detection label used. All markers were introduced once in this way. DAPi, which is excited with UV light and stains nucleic acids, will determine the nuclear morphology of the cells. Control cells will be labeled with CAM5.2 labeled with CY2. Cytokeratins 7, 8, 18 and 19 will be labeled with α-cytokeratin labeled with CY3. An antibody conjugated with CY3.5 will be used to label HER-2 / neu. An antibody conjugated with CY5 will be used to label Muc-1. An antibody conjugated to CY5.5 will be used to label the estrogen receptor. Tumor cells will be identified by using appropriate excitation and emission filters. Again, the use of Celltrax or Cell Spotter ^R cartridge is depicted in the manner described.

  Examples of different types of cancer that can be detected using the compositions, methods and kits of the present invention include Apdoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, cancer, eg, Walker ), Basal cells, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel ) Cells, mucinous, non-small cell lung, oat cell, papillary, skill, bronchiole, bronchiogenic, squamous cell and transitional cell endothelial tumor, melanoma, chondroblastoma, chondroma, Chondrosarcoma, fibroma, fibrosarcoma, giant cell tumor, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma s sarcoma), synovial tumor, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, mesenchyme, midnephroma, myoma, enamel epithelioma, cementoma, odontoma, teratoma, slohobastic (Throphoblastic) tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, columnar tumor, cystadenocarcinoma, cystadenoma, granulosa cell tumor, male germoma (gynandroblastoma), liver cancer, sweat adenoma, islet cell tumor, Leydig Celloma, papilloma, Sertoli cell, follicular cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, rhabdomyosarcoma, rhabdomyosarcoma, ependymoma, ganglion cell, glioma , Medulloblastoma, meningioma, schwannoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, non-chromophilic paraganglioma, antiokeratoma, sclerosing hemangioma (Angioma sclerosing), hemangiomatosis, glomus hemangioma, hemangioendothelioma, hemangioma, angioderma, angiosarcoma Lymphangioma, lymphangiomyoma, lymphangiosarcoma, pineal gland, carcinosarcoma, chondrosarcoma, phyllosarcoma, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphatic vessel Sarcoma, myoma, myxosarcoma, ovarian cancer, rhabdomyosarcoma, sarcoma (Kaposi and mast cells), neoplasm (eg, bone, digestive system, colorectal, liver, pancreas, pituitary gland, testis, orbit, Head and neck, central nervous system, hearing, pelvis, respiratory and urinary), neurofibromatosis and cervical dysplasia.

  The present invention is not limited to the detection of circulating epithelial cells. Endothelial cells were observed in the blood of patients with myocardial infarction. Endothelial cells, cardiomyocytes and virus infected cells, like epithelial cells, have cell type specific determinants that are recognized by available monoclonal antibodies. Thus, the methods and kits of the present invention can be applied to detect such circulating endothelial cells. In addition, the present invention allows the detection of bacterial cells loaded in the peripheral blood of a patient who has an infectious disease and can be evaluated using the compositions, methods and kits of the present invention.

  Some citations for articles, US patents and US patent applications are provided above. The subject matter of each cited reference is fully incorporated by reference throughout this specification and is deemed to be part of this specification.

  Certain preferred embodiments of the present invention are described and specifically exemplified above, but it is not intended that the invention be limited to such embodiments. Various modifications can be made without departing from the spirit of the invention, the full scope of which is set forth in the following claims.

治療の日数およびタイプを図の頂部にて示す。パネル１７Ｃに示す酢酸メゲストールを毎日服用する。ＨＥＲ−２は、処置前にはＣＴＣを発現しない。ＨＥＲ−２陽性への反転により表されるそのＣＴＣの表現型における変化は、ＣＴＣ数の増加として３名全ての患者に明確に検出された。患者番号は表ＸＩＩに言及する。
図１８Ａ−１８Ｃは、疾患進行中のＨＥＲ−２^＋ＣＴＣを持つ患者におけるＣＴＣ上のＨＥＲ−２密度における変動を示す一連のグラフである。ベースラインにてＨＥＲ−２^＋ＣＴＣ、および追跡中に進行した疾患を持つ３名の乳癌患者の処置経過中のＣＴＣ。他の表示は図１７のごとくである。パネル１８Ｂに示したエクセメスタンを毎日服用した。ある割合のＣＴＣが、処置経過中ＨＥＲ−２を発現した。ＣＴＣは、患者２３および２５の処置経過中に実質的に増加した。 図１９Ａ−１９Ｃは、安定な疾患を持つ患者におけるＣＴＣ上のＨＥＲ−２密度における変動を示す一連のグラフである。ベースラインにてＨＥＲ−２^＋ＣＴＣ、および処置中に臨床的に安定したままである疾患を持つ３名の乳癌患者の処置経過中のＣＴＣ。他の表示は図１７のごとくである。アナソトロザロール（パネル１９Ｂ）を毎日服用した。頂部パネル中の患者におけるＣＴＣは処置経過中増加したが、２つの底部パネル中の患者におけるＣＴＣは減少した。 図２０は、本発明の方法を実施するためのプロトコールの例示的概要である。 FIG. 1 is a schematic diagram showing the steps of the sample preparation method of the present invention. 2A-D show various embodiments of the cell spotter ^R chamber of the present invention. Panel 2A: chamber and holder containing two yoked acute magnets; Panel 2B of cells labeled with magnetic nanoparticles randomly placed in a field created by two acute magnets (indicated by dashed lines) E) Computer simulation of orbit. Panel 2C Close-up of the cell trajectory in the Cell Spotter ^R chamber located between the magnets shown in Panel 2B. Panel 2D Top view of chamber surface. The horizontal lines are magnetic nanoparticles lined up along the lines of the strong magnetic field. 3A-D are a series of photomicrographs showing fluorescent images of a frame in a Cell Spotter ^R chamber taken from a blood sample processed from a breast cancer patient. Panel 3A Dapi image showing nuclei from internal controls, leukocytes and tumor cells. Panel 3B DiOC16 image showing fluorescence of 5 control cells. Panel 3C CK-PE image showing fluorescence of two control cells and two candidate tumor cells, one bright and one dimly stained. Panel 3D CD45-APC showing leukocyte fluorescence and no control cell staining, box showing dim PE staining shows APC staining, other box showing no SPC staining confirms it contains CTC To do. FIG. 4 shows the classification of tumor cell candidates. Six columns of thumbnails of tumor cell candidates from breast cancer patients. Columns 201, 202 and 204 are checked to indicate the presence of tumor cells. The thumbnail below the composite image is a composite image of DAPI (purple) and CK-PE staining. L-APC = leukocytes stained with CD45APC, CNTL = control cells stained with DiOC16, EC-PE = epithelial cells stained with cytokeratin-PE, nucleic acid stained with NADYE = DAPI. FIG. 5 shows the results of experiments in which a known number of tumor cells were added to peripheral blood and collected after magnetic selection and analysis by either microscopy (panel A) or flow cytometry (panel B). FIG. 6 shows cells obtained after immunomagnetic cell selection from 10 ml of blood collected from patients with distant metastases of breast cancer taken on days 48, 175 and 300 after the patient entered the study. Flow cytometric analysis of the suspension is shown. After magnetic selection, cells were stained with phycoerythrin (PE) conjugated monoclonal antibody specific for epithelial cells, CD45 PerCP conjugated monoclonal antibody specific for leukocytes and nucleic acid dyes after magnetic selection. Events passing the threshold for nucleic acid dyes required list mode and 85% of the samples were analyzed. Tumor cells are highlighted and shown in black, their numbers are shown in the top right corner; black events consisting of residual white blood cells and debris are shown in gray. FIGS. 7A-H show the number of epithelial cells in 10 ml of blood and the clinical activity of the disease at different time points for 8 patients with active breast cancer. This clinical activity was classified into categories 1 to 4 as described in Table IV. The top bar represents the length of the chemotherapy period. Panel A, respectively, adriamycin (ADR) 90 and 110 mg / ^{m 2,} panel B, ADR30mg / ^{m 2} / week, Vinorelbine (Vin) 20mg / ^{m 2} / week, ADR160mg / ^{m 2} / week, ADR20mg / ^{m 2} / week , panel C, vincristine (Vinc) 0.7mg / ^{m 2} / week, methotrexate (MTX) 30mg / ^{m 2} / week, panel D, vincristine (Vinc) 7mg / ^{m 2} / week, ADR20mg / ^{m 2} / week, Vinb6mg / M ² / week, 5-fluorouracil (5FU) 700 mg / m ² / week. Panel E, Vin 20 mg / m ² / week; 5FU 800 mg / m ² / week + leucovorin 50 mg / m ² / week. Panel F, ifosfamide 18 mg / ^{m 2} / week; 5FU850mg / ^{m 2} / week + leucovorin 35 mg / ^{m 2} / week, 5FU605mg / ^{m 2} / week; Vin20mg / ^{m 2} / week + leucovorin 30 mg / ^{m 2} / week. Panel G, Vin 20 mg / m ² / week, Panel H, Vin 20 mg / m ² / week 8A-8H are a series of photomicrographs showing the results obtained after analysis of immunomagnetically selected cells from the peripheral blood of patients with a history of breast cancer. Panel A, cells from patients 3 years after surgery (T2N1M0), positively stained for cytokeratin. Panel B, cells from a patient 8 years after fully reduced surgery stained with Wright Giemsa (T2N1M1). Panels C and D Cells from a 2-year-old patient stained with Wright Giemsa (T2N0M0). The image was taken with a Pixela digital camera with a 100x objective lens. 9A-9C are a series of graphs showing the correlation between disease severity and the number of circulating cells in three patients with prostate cancer. FIGS. 10A-10H show the CTC and PSA levels measured at 0, 1, 2, 7, 12, 17, and 25 week intervals in the blood of 8 patients with CAP. No significant changes in the clinical activity of the disease during that time course were shown in these patient samples (AD). However, disease activity was increased in these patient samples (E-H). The correlation coefficient between CTC count and PSA level for the patient sample in FIG. 10E is 0.42; for the patient sample in FIG. 10F it is 0.87; for the patient sample in FIG. 0.65; for the patient sample in FIG. 10H it was 0.98. The bar at the top of the panel shows the hormone treatment received. In the patient sample in FIG. 10B, the CTC count never rose above the 99% confidence level of the control group. FIGS. 11A and 11B show CTC and PSA levels measured at intervals of 0, 1, 2, 7, 12, 17, and 25 weeks in the blood of two patients with CAP. The top bar shows the hormone treatment received. Both patients receive chemotherapy; the drug administered and the time of administration are indicated by arrows at the bottom of the figure. FIG. 12 is a graph showing that the number of circulating epithelial cells in patients with colorectal cancer is significantly reduced after surgical removal of the recipient. FIG. 13 is a graph showing that the number of circulating epithelial cells in patients with metastatic disease of the colon increases with the severity and extent of metastatic disease. FIG. 14 is a schematic diagram showing the progression of cancer from early tumor to metastatic growth. Figures 15A-15D show the levels of CTC and HER-2 ⁺ -CTC in blood as measured by flow cytometry analysis. Multiparameter flow cytometric analysis of EpCAM ferrofluid selected cells from 5 ml of blood obtained from patients with breast cancer. Leukocytes and beads are represented as small black dots and their location is indicated in the panel. The gray dot represents a wreckage. The CTC is a large black dot, and the reference used to identify the CTC is indicated by the area in each panel. In the cytokeratin-versus-HER-2 correlation display, the boundaries where cells express excess HER-2 are indicated by dashed lines. FIGS. 16A-D are histograms and scatter plots showing the quantification of HER-2 and CTC on the cell lines of three breast cancer patients. Panel 16A HER-2 expression of leukocytes, PC3 cells and SKBR-3 cells immunomagnetically selected from 5 ml of blood and gated for CD45 and cytokeratin expression. HER-2 expression levels were subdivided into four categories (-, +, ++, +++) based on quantitative assessment of HER-2 expression on PC3 and SKBR-3 cells. (−) No expression, less than 5,000 receptors (WBC), (+) expression between 5,000 and 50,000 receptors (PC-3), (++) 50,000 and 500 Expression between 1,000 receptors and (++++) expression of more than 500,000 receptors (SKBR-3). Panels 16B, 16C and 16D show the expression of cytokeratin and HER-2 on 3 patients from Table XII with breast cancer (2, 20, 15 from CTC. Only CTC is shown in the panel. 17A-17C are a series of graphs showing acquisition of HER-2 overexpression during disease progression. HER-2 ^- CTC at baseline and CTC during the course of treatment of 3 breast cancer patients with disease that progressed during follow-up. The bar indicates the total number of CTCs at each time point. Within each bar, the number of CTCs expressing different levels of HER-2 is indicated by:

Treatment days and types are indicated at the top of the figure. Megestol acetate shown in panel 17C is taken daily. HER-2 does not express CTC prior to treatment. Changes in the CTC phenotype, represented by reversal to HER-2 positivity, were clearly detected in all three patients as an increase in the number of CTCs. Patient numbers refer to Table XII.
18A-18C are a series of graphs showing the variation in HER-2 density on CTC in patients with HER-2 ⁺ CTC during disease progression. HER-2 ⁺ CTC at baseline and CTC during the course of 3 breast cancer patients with disease that progressed during follow-up. The other display is as shown in FIG. Exemestane shown in panel 18B was taken daily. A percentage of CTCs expressed HER-2 during the course of treatment. The CTC increased substantially during the course of treatment for patients 23 and 25. 19A-19C are a series of graphs showing the variation in HER-2 density on CTC in patients with stable disease. HER-2 ⁺ CTC at baseline and CTC during the course of 3 breast cancer patients with disease that remained clinically stable during treatment. The other display is as shown in FIG. Anasotrozalol (panel 19B) was taken daily. CTCs in patients in the top panel increased during the course of treatment, but CTCs in patients in the two bottom panels decreased. FIG. 20 is an exemplary overview of a protocol for performing the method of the present invention.

Claims (82)

A method for assessing patients for the presence of malignancy, comprising:
a) obtaining a biological specimen from a patient, wherein the specimen comprises a mixed cell population suspected of containing hematopoietic and non-hematopoietic malignant cells;
b) preparing a sample mixed with a detectably labeled ligand in which the biological specimen specifically reacts with malignant cells for substantial exclusion of other sample components;
c) contacting the sample with at least one reagent that more specifically labels the malignant cells;
d) analyzing the sample to determine the presence and number of labeled cells, detecting the cells indicating the presence of malignancy, the higher the number of cells, the greater the severity of malignancy. Wherein said method further comprises evaluation of said labeled cells for changes in at least one tumor constitution associated molecule. The method of claim 1, wherein the intermediate step between steps c) and d) comprises contacting the sample with an agent that detectably labels the hematopoietic cells. 3. The method of claim 2, wherein the assessment of the tumor constitution associated molecule comprises contacting the molecule with a detectably labeled agent that has binding affinity for the molecule. The ligand is coupled to a first detectable label, the reagent is coupled to a second detectable label, the hematopoietic cells are labeled with a third detectable label, and the tumor constitution related 4. The method of claim 3, wherein the molecule is contacted with an agent coupled to a fourth detectable label, wherein the first, second, third and fourth detectable labels are different. The labeled malignant cells are converted into multi-parameter flow cytometry, immunofluorescence microscopy, laser scanning cytometry, bright field image analysis, capillary volume analysis, spectroscopic image analysis manual cell analysis, cell spotter ^R analysis, cell track analysis and automatic The method of claim 1, wherein the analysis is by a process selected from the group consisting of cell analysis. Applied to detect and count remaining tumor cells in the biological specimen, wherein the cells are further analyzed for changes in at least one predetermined tumor constitution related molecule after at least one tumor eradication procedure. The method of claim 1 wherein: The biological specimen is periodically obtained from the patient and evaluated for the presence and number of circulating tumor cells, the cells being associated with at least one predetermined tumor constitution as an indicator of the malignant progression 2. The method of claim 1 further analyzed for changes in the molecule. 2. The method of claim 1, wherein the labeled malignant cells are contacted with a chemotherapeutic agent to assess their sensitivity. The sample is an immunomagnetic sample comprising the biological specimen mixed with magnetic particles coupled to the ligand, wherein the immunomagnetic sample is prepared and the immunomagnetic sample is contacted with at least one reagent 2. The method of claim 1 comprising subjecting said immunomagnetic sample to a magnetic field as an intermediate step between allowing to produce a malignant cell suspension enriched as an immunomagnetic sample. . 10. The method of claim 9, wherein the amount of the immunomagnetic sample containing the enriched malignant cells is reduced relative to the amount of the original biological specimen. The method of claim 9, wherein the magnetic particles are colloids. The ligand is a monoclonal antibody specific for at least one cancer cell determinant, and the at least one reagent is on at least one additional monoclonal antibody specific for a second cancer cell determinant and on a non-tumor cell A cell-specific dye is added to the labeled cancer cell-containing fraction, so that cells and cell debris free from nuclei from the analysis are added. 10. The method of claim 9, comprising allowing exclusion. 13. The method of claim 12, wherein the ligand specifically binds to an epithelial cell adhesion molecule on the malignant cell. 13. The method of claim 12, wherein the one or more reagents specifically bind to intracellular cytokeratin. The method according to claim 1, wherein the tumor constitution-related molecule is at least one molecule described in Table XII. The altered tumor constitutional molecule is a protein present on an individual cell, and is selected from the group consisting of cell spotter ^R cytometry, cell track sccytometry, immunofluorescence, mass spectrometry and flow cytometry. The method of claim 1, wherein the method is evaluated. The method of claim 1, further comprising isolation and culture of malignant cells isolated from the biological specimen. 18. The method of claim 17, wherein the cultured malignant cells are grown from a single cell colony to produce a clonal population of the malignant cells. 19. The method of claim 18, wherein the cells of the clonal population are evaluated for changes in tumor constitution related molecules. A malignant cell isolated from a cultured cell obtained from the method of claim 17. A tumor vaccine comprising the cell according to claim 20 or a component thereof. 20. An altered constitution related molecule isolated from the cell of claim 19. A tumor vaccine comprising the altered constitutional molecule according to claim 19 or a fragment thereof. 18. The method of claim 17, further comprising contacting the cell with a therapeutic agent and assessing its sensitivity. The protein tumor constitution-related molecule present in the cultured cells is analyzed by a method selected from the group consisting of protein gel electrophoresis, column chromatography, HPLC, FPLC, immunohistochemistry, histochemistry and Western blot. 23. A method according to claim 22, characterized in that The altered tumor constitutional molecule is a nucleic acid and is evaluated by at least one method selected from the group consisting of nucleic acid library amplification, polymerase chain reaction, agarose gel electrophoresis, Southern blot and Northern blot. 2. A method according to claim 1 characterized in that: 27. The method of claim 26, wherein the library amplification is performed on genetic material isolated from individual cells, and the amplified genetic material is subjected to gene specific probing. 27. The method of claim 26, wherein the library amplification is performed with genetic material isolated from a plurality of malignant cells, and the amplified genetic material is subjected to gene specific probing. A method of assessing a patient for the presence of non-hematopoietic malignancy, comprising:
a) obtaining a biological specimen from a patient, wherein the specimen comprises a mixed cell population comprising hematopoietic and non-hematopoietic malignant cells;
b) preparing an immunomagnetic sample in which the biological specimen is mixed with malignant particles coupled to a ligand that specifically reacts with malignant cells for substantial exclusion of other components;
c) separating the magnetic particles containing malignant cells from non-malignant particle hematopoietic cells; and d) analyzing the magnetic particle-containing cells to determine the presence and number of malignant cells in the sample, Including detecting the cells to be shown and increasing the number of cells, the greater the severity of the malignancy; further comprising evaluating the magnetic particle-containing malignant cells for changes in tumor constitution-related cell molecules The method characterized by the above-mentioned. The altered tumor constitutional molecule is a nucleic acid and is evaluated by at least one method selected from the group consisting of nucleic acid library amplification, polymerase chain reaction, agarose gel electrophoresis, Southern blot and Northern blot. 30. The method of claim 29, wherein: 31. The method of claim 30, wherein the library amplification is performed on genetic material isolated from individual cells, and the amplified genetic material is subjected to gene specific probing. 31. The method of claim 30, wherein the library amplification is performed with genetic material isolated from all malignant cells, and the amplified genetic material is subjected to gene specific probing. 2. The method of claim 1, wherein the altered tumor constitution-related molecule is a carbohydrate and is assessed by a method selected from the group consisting of mass spectrometry, lectin chromatography and boronate affinity. The patient has been diagnosed with epithelial cell cancer selected from the group consisting of prostate cancer, breast cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, head and neck cancer, pancreatic cancer and lung cancer. The method according to 1. 35. The method of claim 34, wherein the labeled cells are evaluated for changes in a tumor constitution associated molecule selected from the group consisting of estrogen receptor, progesterone receptor, Her2 / neu and MUC1. 35. The method of claim 34, wherein the labeled cells are evaluated for changes in a tumor constitution associated molecule selected from the group consisting of androgen receptor, PSA, uPA and PSMA. 35. The method of claim 34, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of thymidylate synthase, EGFR, MUC2 and CEA-15. 35. The method of claim 34, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of CEA-19-3, HCG, MDR and MUC1. 35. The method of claim 34, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of thymidylate synthase, MDR, Her-2 / neu and EGFR. . 2. The method of claim 1, wherein the patient has stage I cancer. 2. The method of claim 1, wherein the patient has stage II cancer. 2. The method of claim 1, wherein the patient has stage III cancer. 2. The method of claim 1, wherein the patient has stage IV cancer. The patient is diagnosed with an epithelial cell carcinoma selected from the group consisting of prostate cancer, breast cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, head and neck cancer, pancreatic cancer and lung cancer. Item 30. The method according to Item 29. 46. The method of claim 45, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of estrogen receptor, progesterone receptor, Her2 / neu and MUC1. 46. The method of claim 45, wherein the labeled cells are evaluated for changes in a tumor constitution associated molecule selected from the group consisting of androgen receptor, PSA, uPA and PSMA. 46. The method of claim 45, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of thymidylate synthase, EGFR, MUC2 and CEA-15. 46. The method of claim 45, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of CEA-19-3, HCG, MDR and MUC1. 46. The method of claim 45, wherein the labeled cells are evaluated for changes in a tumor constitutional molecule selected from the group consisting of thymidylate synthase, MDR, Her-2 / neu and EGFR. . 30. The method of claim 29, wherein the patient has stage I cancer. 30. The method of claim 29, wherein the patient has stage II cancer. 30. The method of claim 29, wherein the patient has stage III cancer. 30. The method of claim 29, wherein the patient has stage IV cancer. As a means of predicting the efficacy of treatment, a method of determining changes in tumor constitution related molecules,
a) obtaining a sample from the patient;
b) if present, isolating and counting circulating tumor cells from the sample;
c) The method comprising measuring the number of at least one predetermined tumor constitution-related molecule on individual cells present in the sample as a means of predicting the efficacy of treatment. As a means of assessing an appropriate dose for treatment, a method for determining a change in a tumor constitutional molecule, comprising:
a) obtaining a sample from the patient;
b) if present, isolate and count circulating tumor cells from the sample; and c) at least one on each individual cell present in the sample as a means of assessing an appropriate dose for treatment. Measuring a predetermined number of tumor constitution related molecules. As a means of monitoring the efficacy of treatment, a method of determining changes in tumor constitution related molecules,
a) obtaining a sample from the patient;
b) if present, isolate and count circulating tumor cells from the sample; and c) as a means of monitoring the efficacy of treatment, as a means of monitoring at least one pre-determined individual cell present in the sample. The method comprising measuring a determined number of molecules related to a tumor constitution. 58. The method of claim 57, wherein the sample is obtained from the patient before, during or after administration of a therapeutic agent. 58. The method of claim 57, further comprising contacting the cell with a therapeutic agent and assessing its sensitivity. 56. The patient according to claim 55, wherein the patient has breast cancer, the tumor constitution related molecule is Her-2 / neu, and the treatment is administration of an anti-Her-2 / neu antibody or a fragment thereof. Method. 57. The patient according to claim 56, wherein the patient has breast cancer, the tumor constitutional molecule is Her-2 / neu, and the treatment is administration of an anti-Her-2 / neu antibody or fragment thereof. Method. 58. The patient according to claim 57, wherein the patient has breast cancer, the tumor constitution related molecule is Her-2 / neu, and the treatment is administration of an anti-Her-2 / neu antibody or a fragment thereof. Method. The patient has breast cancer, the at least one tumor constitutional molecule comprises Her-2 / neu and estrogen receptor, and the treatment is administration of anti-Her-2 / neu antibody or fragment thereof and tamoxifen 56. A method as claimed in claim 55. The patient has breast cancer, the at least one tumor constitution related molecule is Her-2 / neu and estrogen receptor, and the treatment is administration of anti-Her-2 / neu antibody or fragment thereof and tamoxifen 57. The method of claim 56, characterized in that The patient has breast cancer, the at least one tumor constitutional molecule is Her-2 / neu and estrogen receptor, and the treatment is administration of anti-Her-2 / neu antibody or fragment thereof and tamoxifen 58. A method according to claim 57, characterized in that: As a means of assessing cancer progression, a method for determining changes in tumor constitution related molecules,
a) obtaining a sample from the patient;
b) if present, isolating and counting circulating tumor cells from the sample; and c) a predetermined tumor associated with a poor prognosis as a means of assessing cancer progression. Determining the presence of a constitutional related molecule. The tumor constitution related molecule is changed, androgen receptor, cathepsin D, estrogen receptor, estradiol, progesterone receptor, somastatin, steroid receptor coactivator-1 (SRC1), Her-2 (cERB-b), EGFR Ras, c-fos, c-jun, c-myc, p53, p63, nm23 / NDP kinase, PTEN / MMAC1, SMAD4 / DPC4, Notch-1, JAK3, cyclin A, cyclin B, cyclin C, cyclin D, Cyclin E, Ki67, MDR / MRP protein, PSA, prostate acid phosphatase, CA125, CA15-3, CA27-29, HGC, cystic fibrosis transmembrane regulator, laminin receptor, neuron specific Enolase NSE), alphafetoprotein, CD99 / MIC2, DHEA, prolactin, CD66e / CEA, filaggrin, gp200 TAG72 / CA72-4, UPA receptor (CD87), heregulin, IPO-38 thymidylate synthase, topoisomerase Iia, Group consisting of glutathione-S-transferase (GST), lung resistance related protein / major fault protein (LRP / MFP) and 06-methylguanine-DNA methyltransferase (MGMT) 68. The method of claim 66, wherein the method is selected from: The tumor constitution-related molecule is abnormally expressed relative to wild-type expression, androgen receptor, cathepsin D, estrogen receptor, estradiol, progesterone receptor, somastatin, steroid receptor coactivator-1 (SRC1), Her-2 (CERB-b), EGFR, ras, c-fos, c-jun, c-myc, p53, p63, nm23 / NDP kinase, PTEN / MMAC1, SMAD4 / DPC4, Notch-1, JAK3, cyclin A, cyclin B , Cyclin C, cyclin D, cyclin E, Ki67, MDR / MRP protein, PSA, prostatic acid phosphatase, CA125, CA15-3, CA27-29, HGC, cystic fibrosis transmembrane regulator, laminin receptor, neuron specific Enolase (NSE , Alphafetoprotein, CD99 / MIC2, DHEA, prolactin, CD66e / CEA, filaggrin, gp200 TAG72 / CA72-4, UPA receptor (CD87), heregulin, IPO-38, thymidylate synthase, topoisomerase Iia, glutathione 67. The method according to claim 66, wherein the protein is selected from the group consisting of -S-transferase (GST), lung resistance-related protein / major deletion protein (LRP / MFP) and 06-methylguanine-DNA methyltransferase (MGMT). the method of. 68. The method of claim 67, wherein the tumor constitutional molecule is a nucleic acid. 69. The method of claim 68, wherein the tumor constitutional molecule is a nucleic acid. 68. The method according to claim 67, wherein the tumor constitutional molecule is a protein. 69. The method according to claim 68, wherein the tumor substance-related molecule is a protein. A method of performing a whole body biopsy on a patient,
a) obtaining a blood sample from the patient;
b) if present, isolate and count non-hematopoietic tumor cells circulating from the sample; and c) analyze the cells for the presence and number of a predetermined panel of tumor constitution related molecules. The method comprising the steps of: The tumor constitution-related molecule is an androgen receptor, cathepsin D, estrogen receptor, estradiol, progesterone receptor, somastatin, steroid receptor coactivator-1 (SRC1), Her-2 (cERB-b), EGFR, ras , C-fos, c-jun, c-myc, p53, p63, nm23 / NDP kinase, PTEN / MMAC1, SMAD4 / DPC4, Notch-1, JAK3, cyclin A, cyclin B, cyclin C, cyclin D, cyclin E , Ki67, MDR / MRP protein, PSA, prostatic acid phosphatase, CA125, CA15-3, CA27-29, HGC, cystic fibrosis transmembrane regulator, laminin receptor, neuron specific enolase (NSE), alpha fetoprotein CD99 / MIC2, DHEA, prolactin, CD66e / CEA, filaggrin, gp200 TAG72 / CA72-4, UPA receptor (CD87), heregulin, IPO-38, thymidylate synthase, topoisomerase Iia, glutathione-S-transferase (GST) 75. at least two molecules selected from the group consisting of lung resistance-related protein / major deletion protein (LRP / MFP) and 06-methylguanine-DNA methyltransferase (MGMT) Method. 74. The method of claim 73, wherein the patient has epithelial cells selected from the group consisting of prostate cancer, breast cancer, colon cancer, bladder cancer, ovarian cancer, kidney cancer, uterine cancer, head and neck cancer, pancreatic cancer and gastric cancer. A method for identifying changes in circulating tumor cells relative to cells present in a tumor population in situ, comprising:
a) obtaining a biopsy specimen of the tumor population from a patient;
b) if present, isolating circulating tumor cells from the patient;
c) contacting the specimen and the circulating tumor cells with a panel of reagents that detect a plurality of tumor constitution related molecules;
d) detecting any tumor constitution-related molecule present in the circulating tumor cells and in the specimen; then e) the tumor constitution-related molecule is circulating in the biopsy specimen Determining the change in the cell. A test kit for screening patient samples for the presence of non-hematopoietic tumor cells,
a) Coated magnetic nanoparticles comprising a magnetic core material, a protein-based coating material, and an antibody that specifically binds to the first unique determinant of the malignant cell, wherein the antibody is attached to the base coating material Coupling directly or indirectly;
b) at least one antibody having binding specificity for a second unique determinant of the malignant cell;
c) a cell-specific dye for excluding sample components other than the malignant cells from the analysis;
d) a device selected from the group consisting of a Cell Spotter ^R cartridge or Celltrax cartridge; and e) at least one detectably labeled agent having binding affinity for a tumor constitution related molecule. 49. The kit of claim 48, wherein the kit further comprises an antibody having binding affinity for non-target cells, a biological buffer, a permeabilization buffer, a protocol, and optionally an information sheet. The at least one detectably labeled agent having binding affinity for the tumor constitutional molecule is MUC-1, estrogen, progesterone receptor, cathepsin D, p53, urokinase-type plasminogen activator, epidermal growth factor, epithelium 78. A method for screening a patient for breast cancer selected from the group consisting of a growth factor receptor, BRCA1, BRCA2, CA27.29, CA15.5, prostate specific antigen, plasminogen activator inhibitor and Her2-neu. Kit. At least one of the detectably labeled agents having binding affinity for the tumor constitutional molecule is a prostate specific antigen, prostate acid phosphatase, thymosin b-15, p53, HPC1-based prostate gene, creatine kinase and prostate 78. The kit of claim 77, for screening patients for prostate cancer selected from the group consisting of specific membrane antigens. At least one of the detectably labeled agents having binding affinity for the tumor constitutional molecule is carcinoembryonic antigen, C protein, APC gene, p53, thymidylate synthase and matrix metalloproteinase (MMP-9) 78. The kit of claim 77 for screening a patient for colorectal cancer selected from the group consisting of: The group wherein the at least one detectably labeled agent having binding affinity for the tumor constitutional molecule comprises a nuclear matrix protein (NMP22), a bird bladder tumor antigen (BTA) and a fibrinolysis product (FDP) 78. A kit according to claim 77 for screening patients for bladder cancer selected from. 78. The test kit of claim 77, wherein the at least one antibody comprises a panel of antibodies each having binding specificity for a different cancer cell specific determinant.

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