WO2003065042A1 - Methodes et reactifs destines a l'isolement rapide et efficace de cellules cancereuses circulantes - Google Patents

Methodes et reactifs destines a l'isolement rapide et efficace de cellules cancereuses circulantes Download PDF

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Publication number
WO2003065042A1
WO2003065042A1 PCT/US2002/005233 US0205233W WO03065042A1 WO 2003065042 A1 WO2003065042 A1 WO 2003065042A1 US 0205233 W US0205233 W US 0205233W WO 03065042 A1 WO03065042 A1 WO 03065042A1
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cells
tumor
cancer
cell
patient
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PCT/US2002/005233
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English (en)
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Leon W. M. M. Terstappen
Galla Chandra Rao
Shawn Mark O'hara
Paul A. Liberti
Steven Gross
Gerald Doyle
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Immunivest Corporation
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Priority to JP2003564585A priority Critical patent/JP2005516217A/ja
Priority to BR0207290-4A priority patent/BR0207290A/pt
Priority to KR10-2003-7010790A priority patent/KR20030080002A/ko
Priority to AU2002306561A priority patent/AU2002306561A2/en
Priority to CN02807927.2A priority patent/CN1871517A/zh
Priority to IL15725602A priority patent/IL157256A0/xx
Priority to EP02806645A priority patent/EP1360496A4/fr
Priority to CA002438112A priority patent/CA2438112A1/fr
Publication of WO2003065042A1 publication Critical patent/WO2003065042A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/549—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic with antigen or antibody entrapped within the carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/561—Immunoelectrophoresis
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to the fields of oncology and diagnostic testing.
  • the invention is useful for cancer screening, staging, monitoring for chemotherapy treatment responses, cancer recurrence or the like. More specifically, the present invention provides reagents, methods and test kits that facilitate analysis and enumeration of tumor cells, or other rare cells isolated from biological samples.
  • the invention also provides materials and methods for assessing tumor diathesis associated molecules, such as nucleic acids, proteins and carbohydrates, thereby aiding the clinician in the design therapeutic treatment strategies .
  • metastases multiple widespread tumor colonies established by malignant cells that detach themselves from the original tumor and travel through the body, often to distant sites. If a primary tumor is detected at an early stage, it can often be eliminated by surgery, radiation, or chemotherapy or some combination of these treatments. Unfortunately, metastatic colonies are frequently more difficult to detect and eliminate and it is often impossible to treat all of them successfully. Therefore, from a clinical point of view, metastasis can be considered the penultimate event in the natural progression of cancer. Moreover, the ability to metastasize is the property that uniquely characterizes a malignant tumor.
  • Cancer metastasis comprises a complex series of sequential events. These are:
  • PSA Prostate Specific Antigen
  • a useful diagnostic test needs to be very sensitive and reliably quantitative. If a blood test can be developed where the presence of a single tumor cell can be detected in 1ml of blood, that would correspond on average to 3000-4000 total cells in circulation. Innoculum studies for establishing tumors in animals show that injection of 3000-4000 of cells can indeed lead to the establishment of a tumor. Further if 3000-4000 circulating cells represent 0.01% of the total cells in a tumor, then it would contain about 4 x 10 7 total cells. A tumor containing that number of cells would not be visible by any technique currently in existence. Hence, if tumor cells are shed in the early stages of cancer, a test with the sensitivity mentioned above would detect the cancer.
  • Magnetic particles can be classified on the basis of size; large (1.5 to about 50 microns), small (0.7-1.5 microns) , or colloidal ( ⁇ 200nm) , which are also referred to as nanoparticles.
  • Magnetic particles of the type described above are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics.
  • biofunctional polymers e.g., proteins
  • Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, US Patent Nos . 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4 , 659 , 678. Certain of these particles are disclosed to be useful solid supports for immunological reagents . The efficiency with which magnetic separations can be done and the recovery and purity of magnetically labeled cells will depend on many factors.
  • these include : the number of cells being separated, the receptor or epitope density of such cells, the magnetic load per cell, the non-specific binding (NSB) of the magnetic material, • the carry-over of entrapped non-target cells, the technique employed, the nature of the vessel, the nature of the vessel surface, the viscosity of the medium, and • the magnetic separation device employed.
  • a system with 0.8% NSB that recovers 80% of a population which is at 0.25% in the original mixture will have a purity of 25%.
  • the initial population was at 0.01% (one target cell in 10 ⁇ bystander cells), and the NSB were 0.001%, then the purity would be 8%.
  • Extremely low non-specific binding is required or advantageous to facilitate detection and analysis of rare cells, such as epithelial derived tumor cells present in the circulation.
  • high gradient magnetic separation with an external field device employing highly magnetic, low non-specific binding, colloidal magnetic particles is the method of choice for separating a cell subset of interest from a mixed population of eukaryotic cells, particularly if the subset of interest comprises but a small fraction of the entire population.
  • Such materials because of their diffusive properties, readily find and magnetically label rare events, such as tumor cells in blood.
  • the magnetic particles must be specific for epitopes that are not present on hematopoeitic cells.
  • tumor cells Once tumor cells are identified in circulation, it is desirable to further characterize the isolated cells phenotypically or biochemically. Thus, particular tumor diathesis associated molecules, such as nucleic acid molecules, proteins, or carbohydrates that are associated with the malignant phenotype may be analyzed.
  • methods are provided for measuring the level of expression of predetermined tumor diathesis associated molecules present in or on tumor cells identified in the circulation to assist the clinician in diagnosing the type of cancer and assessing the efficacy of chemotherapeutic intervention strategies.
  • a method for assessing a patient for the presence of a malignancy entails obtaining a biological specimen from a patient comprising a mixed cell population suspected of containing hematopoietic and non-hematopoietic malignant cells.
  • a sample is then prepared wherein the biological specimen is mixed with a detectably labeled ligand which reacts specifically with the malignant cells, to the substantial exclusion of other sample components.
  • the sample is contacted with at least one reagent which also specifically labels said malignant cells.
  • the method further comprises assessment of said labeled cells for alterations in at least one tumor diathesis- associated molecule. In one embodiment, this assessment comprises contacting said molecule with a detectably labeled agent having binding affinity therefore.
  • Tumor diathesis associated molecules may be proteins, nucleic acids or carbohydrates and are assessed using conventional methods.
  • malignant cells are analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis manual cell analysis, Cell Spotter® analysis, Cell Tracks analysis and automated cell analysis.
  • the method of the invention may be used to assess residual cancer cell in circulation following medical, radiation or surgical treatment to eradicate the tumor.
  • the method may also be performed periodically over the course of years to assess the patient for the presence and number or tumor cells in the circulation, and alterations in tumor diathesis molecules therein as an indicator of occurrence, recurrence and/or progression of disease.
  • An exemplary method comprises obtaining a sample from a patient; isolating and enumerating circulating malignant cells from said sample if present, and determining the number of at least one predetermined tumor diathesis associated molecule on individual cells present in said sample as a means to predict efficacy of therapy.
  • Such methods may also be used to advantage to assess the appropriate dosage of a given therapeutic regimen and/or for monitoring the efficacy of therapy over time.
  • the methods of the invention provide a "whole body" biopsy based on a simple blood test.
  • methods for culturing tumor cells isolated from the circulation are provided. Such cells may then be contacted with therapeutic agents to assess their sensitivity thereto. Such cells also provide a source for tumor diathesis associated molecules which may or may not be altered.
  • the present invention also encompasses tumor cells or cultures thereof, isolated from the circulation.
  • tumor vaccines derived from the isolated circulating tumor cells of the invention are disclosed.
  • Such tumor vaccines may comprise circulating tumor cells, fragments thereof or purified tumor diathesis associated molecules.
  • a method for identifying alterations in a circulating tumor cells relative to cells present in a tumor mass in situ comprises obtaining a biopsy specimen of said tumor mass from patient and isolating circulating tumor cells from said patient, if any are present. Both the specimen and the isolated circulating tumor cells are then contacted with a duplicate panel of agents which detect a plurality of tumor diathesis associated molecules, such agents optionally being detectably labeled.
  • kits are provided for screening a patient sample for the presence of a non-hematopoietic malignant cells.
  • An exemplary kit of the invention comprises coated magnetic nanoparticles comprising i) a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said malignant cell, the antibody being coupled, directly or indirectly, to said base coating material; ii) at least one antibody having binding specificity for a second characteristic determinant of said malignant cell; iii) a cell specific dye for excluding sample components other than said malignant cells from analysis; iv) a device selected from the group consisting of a Cell Spotter® cartridge or a Cell Tracks cartridge; and at least one detectably labeled agent having binding affinity for a tumor diathesis associated molecule.
  • Such kits may optionally comprise an antibody which has binding affinity for non-target cells, a biological buffer, a permeabilization buffer, a protocol and optionally, an information sheet.
  • Figure 1 is a schematic diagrams showing steps of the sample preparation method of the present invention.
  • Figures 2A-D show various aspects of the CellSpotter® Chamber of the invention.
  • Panel 2A Chamber and holder containing two yoked angular shaped magnets;
  • Panel 2B Computer simulations of trajectories (indicated by the dashed lines) of cells labeled with magnetic nanoparticles placed randomly in a field created by two angular shaped magnets.
  • Panel 2C Close up of the trajectories of the cells within the CellSpotter® Chamber placed in between the magnets as shown in Panel 2B.
  • Panel 2D Top view of the surface of the chamber.
  • the horizontal lines are magnetic nanoparticles capitad up along the ferromagnetic field lines.
  • Figures 3A-3D are a series of micrographs showing fluorescent images of a frame in a CellSpotter® chamber taken from a blood sample processed from a breast cancer patient.
  • Panel 3A Dapi image showing the nuclei from the internal control, leukocytes and tumor cells.
  • Panel 3B DiOCl ⁇ image showing the fluorescence of 5 control cells.
  • Panel 3C CK-PE image showing the fluorescence of 5 control cells and two candidate tumor cells, one bright and one dimly staining.
  • Panel 3D CD45-APC showing the fluorescence of leukocytes and showing no staining of the control cells, the box showing dim PE staining shows APC staining, and the other box showing no APC staining confirming that it contains a CTC.
  • Figure 4 shows the classification of tumor cell candidates.
  • Thumbnails under Composite are composites of DAPI (purple) and CK-PE staining.
  • L-APC leukocyte staining with CD45 APC
  • CNTL control cell staining with DiOCl ⁇
  • EC-PE epithelial cell staining with cytokeratin-PE, NADYE - nucleic acid staining with DAPI.
  • Figure 5 shows the results of model experiments in which known number of tumor cells are spiked into peripheral blood and retrieved after immunomagnetic selection and analysis by either microscopy (Panel A) or flowcytometry (Panel B) .
  • Figure 6 shows flowcytometric analysis of cell suspensions obtained after immunomagnetic cell selection from 10 ml of blood from a patient having distant metastasis of carcinoma of the breast, drawn 48, 175 and 300 days after this patient entered the study.
  • the cells were stained with an epithelial cell specific phycoerythrin (PE) conjugated monoclonal antibody, a leukocyte specific CD45 PerCP conjugated monoclonal antibody and a nucleic acid dye. Events passing a threshold on the nucleic acid dye were acquired into listmode and 85% of the sample was analyzed.
  • the tumor cells are highlighted and illustrated in black and their number is shown in the top right corner; the background events, consisting of residual leukocytes and debris, are illustrated in gray.
  • PE epithelial cell specific phycoerythrin
  • Figure 7A-H shows epithelial cell number in 10 ml of blood and clinical activity of the disease at different time points for eight patients with active carcinoma of the breast.
  • the clinical activity of the disease was classified in categories 1 through 4, as set out in Table IV.
  • the bars at the top represent the length of time of chemotherapy.
  • Panel A adriamycin (ADR) 90 and 110 mg/m 2 respectively
  • Panel B ADR 30 mg/m 2 /week, Vinorelbine (Vin) 20 mg/m 2 /week, ADR 160 mg, ADR 20 mg/m 2 /week
  • Panel C vincristine (Vine) 0.7 mg/m 2 /week, methotrexate (MTX) 30 mg/m/week
  • Panel D vinblastine (Vinb) 7 mg/m 2 /week, ADR 20 mg/m 2 /week, Vinb 6 mg/m/week, 5-fluoruracil (5FU) 700 mg/m 2 /week.
  • Vin vincristine
  • MTX methotrexate
  • Panel E Vin 20 mg/m 2 /week; 5FU 800 mg/m 2 /week + Leukovorin 50 mg/m 2 /week.
  • Panel F ifosfamide (IF) 18 mg/m 2 /week; 5FU 850 mg/m 2 /week + Leukovorin 35 mg/m 2 /week, 5FU 605 mg/m 2 /week; Vin 20 mg/m 2 /week+ Leukovorin 30 mg/m 2 /week.
  • Panel G Vin 20 mg/m 2 /week
  • Panel H Vin 20 mg/m 2 /week
  • Figures 8A-8D are a series of micrographs showing the results obtained following analysis of immunomagnetically-selected cells from peripheral blood of patients with a history of breast carcinoma.
  • Panel A cells from a patient three years after surgery (T2N1M0) staining positive for cytokeratin.
  • Panel B cell from a patient eight years after surgery (T2N1M1) in complete remission stained with Wright Giemsa.
  • Figures 9A-9C are a series of graphs showing the correlation between severity of disease and circulating epithelial cell number in three patients with prostate cancer .
  • Figure 10A-10H show CTC and PSA levels measured at intervals of 0, 1, 2, 7, 12, 17, and 25 weeks in the blood of 8 patients with CAP. No significant change in the clinical activity of the disease during the time course was noted in these patient samples (A-D) . However, disease activity increased in these patient samples (E- H) .
  • the correlation coefficient R between the CTC count and PSA level for the patient sample in Fig. 10E was
  • Figures 11 A and 11 B are a pair of graphs showing
  • Figure 12 is a graph that shows that circulating epithelial cell number in patients with colon cancer is significantly decreased after surgical removal of the tumor .
  • Figure 13 is a graph that shows that circulating epithelial cell number in patients with metastatic disease of the colon increases with the severity and extent of metastatic disease.
  • Figure 14 is a schematic diagram showing the progression of cancer from a primary tumor to growing metastases.
  • Figures 15A-15D are four scatter plots showing the levels of CTCs and HER-2 + -CTC in blood as determined by flowcytometric analysis.
  • Leukocytes and beads are presented as small black dots and their positions are indicated in the panels.
  • the gray dots represent debris.
  • CTCs are the large black dots and the criteria used to identify CTC are indicated by the regions in each of the panels.
  • the border above which cells express HER-2 is indicated by a dashed line.
  • Figures 16A-D are a histogram and scatter plots showing quantification of HER-2 density on cell lines and CTCs of 3 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 on CD45 and cytokeratin expression.
  • the expression levels of HER-2 were subdivided into four categories (-, +, ++, +++) , based on the quantitative assessment of HER-2 expression on PC3 and SKBR-3 cells.
  • Panels 16B, 16C and 16D shows the expression of cytokeratin and HER-2 on CTCs from three patients (2, 20 and 25 from Table XII with breast cancer. Only the CTCs are shown in the panels.
  • Figures 17A-17C are a series of graphs showing acquisition of HER-2 overexpression during disease progression.
  • the bars indicate the total number of CTCs at each time point. Within each bar, the number of CTCs that expressed different levels of HER-2 is indicated by
  • Figures 18A-18C are a series of graphs showing fluctuation in HER-2 density on CTCs in patients with
  • HER-2 + CTCs during disease progression. CTCs during the course of treatment of three breast cancer patients with HER-2 + CTCs at baseline and whose diseases progressed during follow up. Other indicators as per Figure 17. Exemestane shown in Panel 18B was taken daily. A portion of the CTCs expressed HER-2 throughout the course of treatment. CTCs increased substantially during the treatment course of patient 23 and 25.
  • Figures 19A-19C are a series of graphs showing fluctuation in HER-2 density on CTCs in patients with stable disease. CTCs during the course of treatment in three breast cancer patients with HER-2 + CTCs at baseline and whose disease remained clinically stable during therapy. Other indicators as per Figure 17. Anastrozole (Panel 19B) was taken daily. CTCs in the patient in the top panel increased during the treatment course whereas the CTC in the patients in the two bottom panels decreased.
  • Figure 20 is an exemplary schema of a protocol for practicing the methods of the present invention. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides compositions, methods and kits for the rapid and efficient isolation of rare target bioentities from biological samples.
  • the methods described may be used effectively to isolate and characterize tumor cells present in a blood sample while at the same time minimizing the selection of non-specifically bound or entrapped cells.
  • Cancer staging systems describe how far cancer has spread anatomically and attempt to put patients with similar prognosis and treatment in the same staging group.
  • the concept of stage is applicable to almost all cancers except for most forms of leukemia. Since leukemias involve all of the blood, they are not anatomically localized like other cancers, so the concept of staging is often not applied to this type of cancer.
  • a few forms of leukemia do have staging systems which reflect various measures of how advanced the disease is. For most solid tumors, there are two related cancer staging systems, the Overall Stage Grouping, and the TNM system.
  • Stage Groupings (Roman Numeral Staging) system, cases are grouped into four stages denoted by Roman numerals I through IV, or are classified as "recurrent.”
  • stage I or early stage cancers, are small localized cancers that are usually curable, while stage IV usually represents inoperable or metastatic cancer.
  • Stage II and III cancers are usually locally advanced and/or with involvement of local lymph nodes.
  • these stages are defined precisely, but the definition is different for each kind of cancer.
  • it is important to realize that the prognosis for a given stage also depends on what kind of cancer it is, so that a stage II non small cell lung cancer has a different prognosis from a stage II cervical cancer.
  • stages I-IV are actually defined in terms of a more detailed staging system called the "TNM" system.
  • TNM stands for Tumor, Nodes, and Metastases. Each of these is categorized separately and classified with a number to give the total stage.
  • T1N1M0 cancer means the patient has a Tl tumor, Nl lymph node involvement, and no distant metastases.
  • T, N and M are specific to each cancer, but it is possible to broadly define their meaning.
  • T Tumor- Classifies the extent of the primary tumor, and is normally given as TO through T4.
  • TO represents a tumor that has not even started to invade the local tissues. This is called "In situ".
  • T4 represents a large primary tumor that has probably invaded other organs by direct extension, and which is usually inoperable.
  • N Lymph Nodes- Classifies the amount of regional lymph node involvement. It is important to understand that only the lymph nodes draining the area of the primary tumor are considered in this classification. Involvement of distant lymph nodes is considered to be metastatic disease. The definition of just which lymph nodes are regional depends on the type of cancer. NO means no lymph node involvement while N4 means extensive involvement. In general more extensive involvement means some combination of more nodes involved, greater enlargement of the involved nodes, and more distant (But still regional) node involvement.
  • Metastasis- M is either MO if there are no metastases or Ml if there are metastases. As with the overall staging system, the exact definitions for T and N are different for each different kind of cancer.
  • stage II non-small cell lung cancer means a Tl or T2 primary tumor with Nl lymph node involvement, and no metastases (M0) .
  • M0 no metastases
  • the presence of tumor cells in the circulation can be used to screen for cancer in place of, or in conjunction with, other tests, such as mammography, or measurements of PSA.
  • other tests such as mammography, or measurements of PSA.
  • the organ origin of such cells may readily be determined, e.g., breast, prostate, colon, lung, ovarian or other non-hematopoietic cancers.
  • cancer cells can be detected, while there are essentially no clinical signs of a tumor, it will be possible to identify their presence as well as the organ of origin.
  • cancer should be thought of as a blood borne disease characterized by the presence of potentially very harmful metastatic cells, and therefore, treated accordingly.
  • follow-up treatment such as radiation, hormone therapy or chemotherapy is required. Predicting the patient's need for such treatment, or the efficacy thereof, given the costs of such therapies, is a significant and beneficial piece of clinical information.
  • target bioentities refers to a wide variety of materials of biological or medical interest and can be distinguished from “non-target” materials that are present in the specimen. Examples include hormones, proteins, peptides, lectins, oligonucleotides, drugs, chemical substances, nucleic acid molecules, (e.g., RNA and/or DNA) and particulate analytes of biological origin, which include bioparticles such as cells, viruses, bacteria and the like.
  • rare cells such as fetal cells in maternal circulation, or circulating cancer cells may be efficiently isolated from non-target cells and/or other bioentities, using the compositions, methods and kits of the present invention.
  • biological specimen includes, without limitation, cell-containing bodily fluids, including without limitation, peripheral blood, tissue homogenates, nipple aspirates, colonic lavage, sputum, bronchial lavage, and any other source of cells that is obtainable from a human subject.
  • An exemplary tissue homogenate may be obtained from the sentinel node in a breast cancer patient.
  • determinant when used in reference to any of the foregoing target bioentities, refers broadly to chemical mosaics present on macromolecular antigens that often induce an immune response. Determinants may also be used interchangeably with “epitopes” .
  • Determinants may be specifically bound by a biospecific ligand or a biospecific reagent, and refers to that portion of the target bioentity involved in, and responsible for, selective binding to a specific binding substance (such as a ligand or reagent) , the presence of which is required for selective binding to occur.
  • determinants are molecular contact regions on target bioentities that are recognized by agents, ligands and/or reagents having binding affinity therefor, in specific binding pair reactions.
  • binding pair includes antigen-antibody, receptor-hormone, receptor- ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin and virus-receptor interactions.
  • Gene specific probing refers to methods wherein nucleic acid molecules which are complementary to tumor diathesis associated molecules are used to detect the presence or absence of such molecules. Such nucleic acids may or may not be detectably labeled.
  • Various other determinant-specific binding substance combinations are contemplated for use in practicing the methods of this invention, and will be apparent to those skilled in the art.
  • tumor diathesis is used herein to refer to a constitutional susceptibility or predisposition to malignant disease. Predisposition or susceptibility to malignant disease may be inherited, or due to somatic cell mutations that lead to dysregulated cellular proliferation.
  • tumor diathesis associated molecule refers to intracellular and extracellular molecules that are altered biochemically or expressed aberrantly as a cell progresses from a normal to malignant phenotype. Such molecules include without limitation, hormones and hormone regulated proteins, oncogenes, tumor suppressor proteins, apoptosis associated molecules, cell cycle and proliferation associated molecules, carbohydrate molecules associated with malignancy, cytoskeletal proteins and proteins involved in maintenance of cell-to- cell contacts.
  • malignant cell refers to a cell which is biochemically and/or phenotypically altered such that normal stringent control of cellular proliferation and/or localization is lost. Malignant cells are not normally present in circulation.
  • antibody includes immunoglobulins, monoclonal or polyclonal antibodies, immunoreactive immunoglobulin fragments such as F(ab), and single chain antibodies (sfV) . Also contemplated for use in the invention are peptides, oligonucleotides or a combination thereof which specifically recognize determinants with specificity similar to traditionally generated antibodies. As mentioned previously, complementary nucleic acids are encompassed within the meaning of "specific binding pair”.
  • detectably label is used to herein to refer to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, is indicative of the presence of the target bioentity in the test sample.
  • useful detectable labels include, but are not limited to the following: molecules or ions directly or indirectly detectable based on light absorbance, fluorescence, reflectance, light scatter, phosphorescence, or luminescence properties; molecules or ions detectable by their radioactive properties; molecules or ions detectable by their nuclear magnetic resonance or paramagnetic properties. Included among the group of molecules indirectly detectable based on light absorbance or fluorescence, for example, are various enzymes which cause appropriate substrates to convert, e.g., from non-light absorbing to light absorbing molecules, or from non-fluorescent to fluorescent molecules .
  • the phrase "to the substantial exclusion of” refers to the specificity of the binding reaction between the biospecific ligand or biospecific reagent and its corresponding target determinant. Biospecific ligands and reagents have specific binding activity for their target determinant yet may also exhibit a low level of nonspecific binding to other sample components.
  • the phrase “early stage cancer” is used interchangeably herein with “Stage I” or “Stage II” cancer and refers to those cancers that have been clinically determined to be organ-confined. Also included are tumors too small to be detected by conventional methods such as mammography for breast cancer patients, or X-rays for lung cancer patients. While mammography can detect tumors having approximately 2 x 10 8 cells, the methods of the present invention should enable detection of circulating cancer cells from tumors approximating this size or smaller.
  • enrichment refers to the process of substantially increasing the ratio of target bioentities (e.g., tumor cells) to non-target materials in the processed analytical sample compared to the ratio in the original biological sample.
  • target bioentities e.g., tumor cells
  • red cells are not counted when assessing the extent of enrichment.
  • circulating epithelial cells may be enriched relative to leucocytes to the extent of at least 2,500 fold, more preferably 5,000 fold and most preferably 10,000 fold.
  • clonal expansion when used in reference to isolated, circulating tumor cells, refers to methods of placing the isolated cells in culture under conditions whereby the cells proliferate. Single cells may be cultured such that they form colonies which may then be clonally expanded to generate a population of essentially homogeneous cancer cells. Portions of such cells or the cells themselves may be used to generate tumor vaccines.
  • tumor vaccine refers to agents that contain a specific protein of the tumor cell that can be used to stimulate an immune response. Vaccines can comprise viruses, small proteins, or whole cells. Methods for generating tumor vaccines using tumor cells infected with an adenovirus-associated vector are disclosed in US Patent 6,171,597. Additional methods for generating tumor vaccines from circulating tumor cells are disclosed in US Patent 5,993,829.
  • the preferred magnetic particles for use in carrying out this invention are particles that behave as colloids. Such particles are characterized by their sub- micron particle size, which is generally less than about 200nm (0.20 microns), and their stability to gravitational separation from solution for extended periods of time. In addition to the many other advantages, this size range makes them essentially invisible to analytical techniques commonly applied to cell analysis. Particles within the range of 90-150 nm and having between 70-90% magnetic mass are contemplated for use in the present invention. Suitable magnetic particles are composed of a crystalline core of superparamagnetic material surrounded by molecules which are bonded, e.g., physically absorbed or covalently attached, to the magnetic core and which confer stabilizing colloidal properties.
  • the coating material should preferably be applied in an amount effective to prevent non-specific interactions between biological macromolecules found in the sample and the magnetic cores.
  • biological macromolecules may include carbohydrates such as sialic acid residues on the surface of non-target cells, lectins, glyproteins, and other membrane components.
  • the material should contain as much magnetic mass per nanoparticle as possible.
  • the size of the magnetic crystals comprising the core is sufficiently small that they do not contain a complete magnetic domain.
  • the size of the nanoparticles is sufficiently small such that their Brownian energy exceeds their magnetic moment. As a consequence, North Pole, South Pole alignment and subsequent mutual attraction/repulsion of these colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, contributing to their solution stability.
  • Magnetic particles should be separable in high magnetic gradient external field separators. That characteristic facilitates sample handling and provides economic advantages over the more complicated internal gradient columns loaded with ferromagnetic beads or steel wool.
  • Magnetic particles having the above-described properties can be prepared by modification of base materials described in U.S. Patents Nos. 4,795,698, 5,597,531 and 5,698,271. Their preparation from those base materials is described below. Malignant tumors are characterized by their ability to invade adjacent tissue. 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 be shed into the circulation in a 24 hour period (Butler, TP & Gullino PM, 1975 Cancer Research 35:512-516).
  • the shedding capacity of a tumor is most likely dependent on the aggressiveness of the tumor. Although tumor cells are shed into the circulation on a continuous basis, it is believed that none or only a small fraction will give rise to distant metastasis (Butler & Gullino, supra) . Using the following assumptions, one can approximate the frequency of tumor cells in circulation as follows:
  • a tumor with a diameter of 1 mm contains 10 7 cells, and 4% or 4 x 10 5 cells will be shed into the circulation in a 24 hour period;
  • the frequency of tumor cells in peripheral blood of a patient with a 1mm diameter tumor is approximately 6 tumor cells/lOOml of blood. Increase in tumor mass might be expected to be proportional to an increase in the frequency of the circulating tumor cells. If this were found to be the case, methods available with this level of sensitivity would facilitate assessment of tumor load in patients with distant metastasis as well as those with localized disease. Detection of tumor cells in peripheral blood of patients with localized disease has the potential not only to detect a tumor at an earlier stage but also to provide indications as to the potential invasiveness of the tumor.
  • leukopheresis products were more likely to contain tumor cells when obtained from individuals with disseminated disease (Brugger et al., 1994, supra) . These studies, however, do not report quantitative data, nor do they report that tumor cells can be found in peripheral blood of patients with localized disease. Given these observations, one may hypothesize that a highly sensitive and quantitative test that counts the number of tumor cells in peripheral blood may be used to determine actual tumor load. To assess the feasibility of such testing, a sensitive cellular assay was developed which allows precise enumeration of circulating carcinoma cells that is limited only by the blood volume to be tested.
  • Cytometry (Compucyte) , bright field base image analysis (Chromavision) , and capillary Volumetry (Biometric Imaging) .
  • the enumeration of circulating epithelial cells in blood using the methods and compositions of a preferred embodiment of the present invention is achieved by immunomagnetic selection (enrichment) of epithelial cells from blood followed by the analysis of the samples by multiparameter flowcytometry .
  • the immunomagnetic sample preparation is important for reducing sample volume and obtaining a 10 4 fold enrichment of the target (epithelial) cells.
  • the reagents used for the multiparameter flowcytometric analysis are optimized such that epithelial cells are located in a unique position in the multidimensional space created by the listmode acquisition of two light scatter and three fluorescence parameters. These include
  • an antibody against the pan-leukocyte antigen, CD45 to identify leucocytes (non-tumor cells) an antibody against the pan-leukocyte antigen, CD45 to identify leucocytes (non-tumor cells) ; 2) a cell type specific or nucleic acid dye which allows exclusion of residual red blood cells, platelets and other non-nucleated events; and 3) a biospecific reagent or antibody directed against cytokeratin or an antibody having specificity for an EpCAM epitope which differs from that used to immunomagnetically select the cells.
  • the method of analysis of the enriched tumor cell population will depend on the intended use of the invention. For example, in screening for cancers or monitoring for recurrence of disease, as described hereinbelow, the numbers of circulating epithelial cells can be very low.
  • Example 11 Since there is some "normal" level of epithelial cells, (very likely introduced during venipuncture) , a method of analysis that identifies epithelial cells as normal or tumor cells is desirable. In that case, microscopy based analyses may prove to be the most accurate. Such examination might also include examination of morphology, identification of known tumor diathesis associated molecules (e.g., oncogenes) . Suitable tumor diathesis associated molecules that may be further analyzed in accordance with the methods of the invention are provided in Example 11.
  • tumor diathesis associated molecules e.g., oncogenes
  • an analytical method that enumerates such cells should be sufficient.
  • the determination of patient status according to the methods described herein is made based on a statistical average of the number of circulating rare cells present in the normal population.
  • Levels of circulating epithelial cells in the early stage cancer patient and in patients with aggressive metastatic cancer can also be statistically determined as set forth herein. 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 Formulation of improved magnetic nanoparticles for the efficient isolation of rare cells from whole blood
  • Rare cells e.g., tumor cells in patients with epithelial derived tumors, fetal cells in maternal blood or the like
  • Rare cells can be present in frequencies below one rare cell per ml of blood.
  • the number of blood smears required to detect such rare cells is prohibitively large.
  • 10 rare cells in 10 ml of blood which corresponds to 10 tumor cells in 5-10 x 10 7 white blood cells (leukocytes)
  • cells can be transferred to a microscope slide by cytocentrifugation or by settling, stained with an antibody specific for the rare cells of interest and read manually or automatically.
  • the maximum number of cells that can be transferred to one slide is about 500,000 cells which means 100-200 slides are required to process 10 ml of blood.
  • enrichment methods such as sample volume reduction and removal of erythrocytes and platelets by density gradient separation or erythrocyte lysis procedures are used for isolating rare cells so as to significantly reduce the number of slides to be analyzed.
  • magnetic enrichment is the preferred method for cell separations and, ideally, the nanoparticles employed for this purpose should not have to be removed prior to analysis. Accordingly, the nanoparticles should be small enough so as not to interfere with analytical measurements, i.e. below about 250 nm.
  • the nanoparticles are below 220 nm so as to make them filter sterilizable.
  • the nanoparticle should be large enough and magnetically responsive enough to permit cell separation from simple laboratory tubes, i.e., test tubes, centrifuge tubes, vacutainers and the like in external gradient magnetic separators. Again, as previously noted internal gradient devices are cumbersome, costly and inefficient for the recovery of rare cells. Also, the nanoparticles and magnetic device should give high and reproducible recovery with low non-specific binding.
  • US Patent No. 5,597,531 describes the synthesis of highly magnetic particles, referred to as direct coated (DC) particles which have many of these characteristics.
  • nanoparticles are composed of quasispherical agglomerates of crystalline magnetite or other magnetic oxides which are coated with polymers or proteins (based coated magnetic particles) . Because of their structure (magnetic core and polymer coat where the core diameter is >>> than the thickness of the coat) they are about 80- 85% magnetic mass. The non-specific bindings of these nanoparticles are in the range of 5-8 % and they are, therefore, not very practical for rare cell separations. Thus if one is enriching cells present at one cell per ml then at 80% capture efficiency, the best result to be expected using 10ml of whole blood (considering leukocytes alone) would be 8 cells recovered in a total of 4 million, i.e. a 16-17 fold enrichment.
  • the magnetic particles described in U.S. Patent 5,597,531 do, however, have the appropriate magnetic properties to perform separations with open field separators and from simple laboratory tubes. Further, their mean size is well under the limit suggested above and, hence, they do not interfere with various analytical procedures. Based on extensive studies with those materials, the major contributing factor to non-specific binding to cells was discovered to be the presence of bare crystalline iron oxides on the nanoparticles due to incomplete coating. Such incompletely coated crystals have a sufficiently high positive charge at physiological pH that they are very likely to bind strongly to biological macromolecules, such as negatively charged sialic acid on cell surfaces. An improved method for making particles is described in U.S. Patent No. 5,698,271.
  • Nanoparticles made with bovine serum albumin (BSA) coating using this process have a 3-5-fold lower non-specific binding characteristic for cells when compared to the DC-BSA materials of US Patent 5,579,531. This decrease in non-specific binding has been shown to be directly due to the increased level of BSA coating material. When such nanoparticles were treated so as to remove BSA coating, non-specific binding returns to high levels. It was thus determined that a direct relationship exists between the amount of BSA coated on iron oxide crystal surfaces and the nonspecific binding of cells.
  • BSA bovine serum albumin
  • base coated magnetic particles could be prepared that were devoid of excessively small or large nanoparticles.
  • base coated particles of mean diameter lOOnm can be produced which contain at best trace amounts of material smaller than 80 nm or over 130 nm.
  • material of about 120 nm can be made with no appreciable material smaller than 90-95 nm and over 160 nm.
  • Such materials performed optimally with regard to recovery and could be made sub-optimal by the inclusion of 60-70 nm nanoparticles.
  • the preferred particle size range for use in practicing this invention is 90-150 nm for base coated magnetic particles, e.g., BSA-coated magnetite.
  • Particles falling within this preferred range may be obtained using the procedure described by Liberti et al . In Fine Particles Science and Technology, 777-90, E. Pelizzetti (ed. ) (1996).
  • Monoclonal antibody specific for rare cells can be directly coupled to, for example, the BSA base coating on the DC magnetic particles by standard heterobifunctional chemistry (referred to herein as direct coupling method) .
  • Heterobiofunctional linkers used for these purposes include sulfosuccinimidil-4- [maleimidomethyl] cyclohexane- 1-carboxylate (SMCC) .
  • biotinylated monoclonal antibodies can be coupled to streptavidin that has been coupled to the base coated particles.
  • This conjugate method is referred to herein as a piggyback method.
  • streptavidin is coupled to the base coated magnetic particles by the same chemistry as the direct coupling method.
  • monobiotinylated antibody is allowed to react with streptavidin magnetic particles for 1 hour and then the remaining streptavidin binding sites quenched with free biotin. It is important to quench the remaining streptavidin sites after antibody coupling to prevent binding of any biotinylated antibody to magnetic particles during isolation of rare cells or the cell analysis step.
  • the surfaces of the magnetic crystals are thus coated more extensively with multiple layers of protein and appear to be sterically "protected” . This prevents binding of non-target cells to the magnetic particles.
  • a limited number of streptavidin binding sites on the magnetic particles are occupied with biotin-antibody and the remainder are saturated with free biotin by the quench process described above.
  • the excess streptavidin binding sites were quenched and saturated with monobiotin-BSA instead of free biotin.
  • the rationale for this approach is that quenching with monobiotin BSA should further sterically inhibit cells from coming in contact with uncoated regions of the nanoparticles, i.e. give better coverage of the nanoparticles. It was shown by carbon analysis that this process increases the amount of protein coupled to the particles.
  • the two magnetic particle preparations were compared in experiments assessing recovery of spiked Colo 205 from whole blood and for non-specific binding of leukocytes. The results are presented in Table II.
  • Monobiotin-BSA may be prepared by conjugating a limited amount of biotin to BSA, such that 30- 40% of the resultant product has no bound biotin.
  • magnetic particles having a homogeneous size distribution and biotin-BSA quenched streptavidin binding sites performed extremely well in the assay methods of the present invention.
  • a good recovery of the spiked epithelial tumor cells and almost an order of magnitude reduction in nonspecific binding is obtained using these particles, compared with the biotin-blocked nanoparticles.
  • these materials and the results obtained with them define a very useful product that can be further optimized.
  • the improved ferrofluid product is made as magnetic as possible, is coated so as to exclude all possible interactions of the magnetic core with any substances in blood including cells (presumably coated with a nonporous monolayer) and are well defined in its size range and distribution. In the preferred situation, a coat material is used which does not interact with biological materials.
  • a means for blocking them is required.
  • a material to be as magnetic as possible those produced as described in US Patent Nos. 5,579,531 and 5,698,271 are preferred starting materials . They are preferable because they are composed of large magnetic cores with an apparent but not complete monolayer of base coating material.
  • the core will be about 90 nm of an appropriate magnetic oxide such as magnetite.
  • Such nanoparticles because of the relative size of the cores and coat material are clearly as magnetic as is possible. This is apparent if one considers that the function of the coating is to keep the nanoparticles from undesired interactions with each other, which would lead to macroscopic agglomeration.
  • the coating also promotes sufficient interactions with solvent molecules so as to maintain colloidal behavior and provides a convenient chemical means for coupling.
  • the nanoparticles of US Patent Nos. 5,579,531 and 5,698,271 are also preferred as a starting material as they have sufficient monolayer coating wherein "holes" in the monolayer can be filled in several ways, viz., sterically and physically.
  • any coating that promotes the effective complete coverage of the magnetic core, so as to inhibit interactions of the core material with blood components or any other non-specific effects in any other system would be suitable. The less mass such a coating might add to the nanoparticles the better, so as to maximize the magnetic mass to nanoparticle mass ratio .
  • a semi-automated system was developed that processes and analyzes 7.5 ml of blood for the presence of epithelial derived tumor cells.
  • Cells of epithelial cell origin are immunomagnetically labeled and separated from blood.
  • the magnetically captured cells are differentially fluorescent labeled and placed in an analysis chamber.
  • Four-color fluorescent imaging is used to differentiate between debris, hematopoeitic cells and circulating tumor cells (CTC) of epithelial origin.
  • An algorithm is applied on the captured images to enumerate an internal control and identify all objects that potentially classify as tumor cell based on size and immunophenotype . Thumbnail images of each object are presented in an user interface from which the user can determine the presence of tumor cells.
  • the internal control showed consistent and reproducible results between systems and operators.
  • CTC were detected in blood samples of patients with metastatic breast cancer, however, other diseases may be analyzed with the system.
  • tumor cells can be present in blood of carcinoma patients at extremely low frequencies ( ⁇ 10 cells per ml) .
  • the laborious manual sample preparation and complex analysis methods involved in detecting the presence of CTC often lead to erroneous results.
  • the root causes of erroneous results can frequently be traced to the cumulative effects of systematic and /or random pre-analytical errors, i.e. errors occurring during sample preparation or pre-processing stages rather than in the analytical method itself.
  • Pre-analytical errors may manifest as variations due to technique-sensitive process steps as well as random or systematic variations from operator to operator.
  • manual sample preparation in rare cell analysis when performed inconsistently, can result in high variability and unreliable assay results.
  • the system is primed with System Buffer (Immunicon Corp, Huntingdon Valley, PA) that is also used in various steps during the procedure.
  • This buffer consists of phosphate buffered saline, EDTA, and proteins to reduce nonspecific binding of reagents to cells and to system components.
  • Magnetic nanoparticles are coupled to monoclonal antibodies (mabs) specific for epithelial cell adhesion molecule (EpCAM) as 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.
  • Streptavidin in AB buffer (System buffer with streptavidin added) is added to the specimens before the addition of CA-EpCAM to form aggregates, which minimize differences in magnetic capture efficiencies of cells with different EpCAM antigen densities.
  • the magnetically captured cells are fluorescently labeled with anti- cytokeratin conjugated to Phycoerythrin (CK-PE) and anti- CD45 conjugated to Allophycocyanin (CD45-APC) in addition to the nucleic acid specific dye DAPI (4 , 6-diamidino-2- phenylindole) .
  • the anti-cytokeratin recognizes keratins 4,6,8,10,13, and 18, present in cells of epithelial origin.
  • the mabs are added into a buffer medium that contains a detergent to permeabilize the cytoplasmic membrane of the cells (Immuniperm) .
  • the final resuspension buffer in Cellfix (Immunicon Corp,
  • Biotin by virtue of its higher affinity for streptavidin, serves to displace desthiobiotin from streptavidin, thereby reversing the controlled cross linking between desthiobiotin on the ferrofluid particles and streptavidin in the aggregates formed in the earlier steps of the assay.
  • Internal control cells are the subject of US Patent Application 09/801,471, the entire disclosure of which is incorporated by reference herein. These control cells can be successfully derived from the cancer tumor cell-lines. As described herein, cells from the breast cancer line, SKBR-3, are stabilized and uniquely labeled with the fluorescent membrane dye DiOCl ⁇ (from Molecular Probes) to permit differentiation from endogenous tumor cells. Approximately 1000 control cells are spiked into each specimen. The control cells express EpCAM and are captured concurrently with the tumor cells. Control cells also express intracellular cytokeratin and staining with CK-PE verifies the quality of this reagent. The percent recovery of added control cells provides an indicator of total reagents and system performance for each specimen, unlike external controls that can detect only systematic errors.
  • DiOCl ⁇ from Molecular Probes
  • the system is equipped with two magnetic separators each consisting of a set of four rectangular rare earth magnets arranged in a quadrupole configuration with a 17mm diameter cavity surrounded by a circular steel yoke.
  • each separator can hold a 15 ml conical tube.
  • Adjacent to each separator is a magnetic yoke that holds the analysis chamber (CellSpotter® chamber) into which the final sample is transferred. This chamber assembly can be removed from the system and placed onto the microscope stage.
  • the tube transport consists of two movable tube arms, for raising and lowering the tubes into and out of the magnetic separators, along with a rotary turntable for positioning the tubes at various process positions.
  • a probe washbowl is mounted on the turntable allowing internal and external washing of the aspiration and transfer probes.
  • a Cavro XE1000 digital syringe pump with 5mL syringe is used for fluid deliveries transfers and probe washing.
  • a Cavro SP Smart Peristaltic pump with PharMed tubing is used for aspirations to waste.
  • Two pinch valves and one 3-way valve (Bio-Chem) are used to control fluid paths.
  • Separate aspiration and transfer probes fabricated from 13 AWG Inconel tubing (non-magnetic) are used for fluid access to the 15 ml conical tubes and the chamber.
  • a through-beam photoelectric sensor (Omron) is used for determining the height of the packed red-cell layer to allow precisely controlled plasma aspiration.
  • the system is controlled using an 8-bit microcontroller running firmware to execute the motion controls, process controls, and the operator interface commands.
  • the protocol itself (incubation times, fluid processing steps, etc.) is encoded into a separate memory chip.
  • the operator interface consists of a four-by-five key keypad, a 2-line by 20-character LCD display, and an audible alarm.
  • the CellSpotter® system utilizes a Nikon E-400 microscope equipped with a 10X objective (WD 4mm, NA 0.45), a high resolution X, Y, Z stage and a filter cube changer control that are controlled with a Ludl MAC2002 controller, which in turn, is controlled by a PC via RS- 232.
  • Excitation, dichroic and emission filters in each of four cubes are for DAPI 365nm/400nm/400nm, for DiOCl ⁇ 480nm/ 495nm/ 510nm, for PE 546nm/ 560nm/ 580nm and for APC 620nm/ 660nm/ 700nm.
  • FIG. 1 The steps involved in preparation of the sample for analysis are depicted in Figure 1.
  • a 15 ml conical tube 7.5 ml of blood, 6 ml of System Buffer and 100 ⁇ l (about 1000) of control cells are added and mixed.
  • the sample is centrifuged at 800g for 10 min and placed onto the system.
  • the system locates the top of the packed red cell layer in the tube and a probe is introduced into the tube to aspirate the plasma without disturbing the buffy coat layer.
  • the tube is taken from the system and 6 ml of AB buffer and 100 ⁇ l of CA-EpCAM ferrofluid are added and mixed.
  • the tube is placed in the system and the sample tube is inserted and withdrawn from the magnetic separator under system control, thereby providing precise control over separation and removal times.
  • the ferrofluids While in the magnetic field the ferrofluids are moving laterally through the blood sample thereby increasing the labeling efficiency of potential target cells as well as moving the magnetically labeled CTC and unbound ferrofluid to the wall of the tube.
  • the probe After incubation and separation for 20 minutes, the probe is slowly lowered into the tube, aspirating and discarding the blood sample to waste.
  • the tube is mechanically moved out of the separator and 3 ml of System Buffer is added to the tube.
  • the collected cells and ferrofluid are resuspended by mixing the tube. The system lowers the tube into the magnet and aspirates uncollected material after 10 minutes.
  • the tube is moved out of the magnet by the system and 200 ⁇ l of Immunoperm and 60 ⁇ l of staining reagents are added and mixed. After incubation for 15 minutes the excess staining reagents are aspirated and discarded by the system and the tube is moved out of the magnet. Ten minutes after 250 ⁇ l Cellfix is added, the system transfers the sample into the chamber. The volume of the chamber is approximately 320 ⁇ l and the system uses 100 ⁇ l of system buffer as a rinse to assure that residual cells in the tube are transferred into the chamber. The chamber is slightly overfilled to avoid air entrapment during capping with the plug seal. After each step in which a probe touches a sample, the probe is thoroughly washed by the system to eliminate any cell or reagent carryover.
  • Figure 2A shows the analysis chamber and the magnet yoke assembly that holds the chamber between the two magnets.
  • a computer program was written to simulate the movement of magnetically labeled cells in the chamber. The objective was to move all magnetically labeled cells to the upper surface of the chamber while preventing movement to the magnet poles. The distance from the chamber surface to the surface of the magnet must also be short enough to permit viewing through a microscope objective.
  • Figure 2B shows such a simulation, the chamber is outlined between the North (N) and South (S) pole of the magnets and the dashed lines indicate the trajectory of magnetically labeled cells.
  • Figure 2C shows a magnification of the trajectory within the chamber.
  • Figure 2D shows the top view of the CellSpotter® chamber from an experiment in which cells and magnetic nanoparticles are introduced into the chamber.
  • the horizontal lines distributed homogeneously over the surface of the chamber represent the magnetic nanoparticles that align along the magnetic field lines.
  • the surface of the chamber is 80.2 mm 2 and has to be scanned completely for any objects staining with DAPI, DiOC16, CK-PE and CD45-APC.
  • the combination of the objective and digital camera results in a pixel size of 0.45 (0.67X0.67) ⁇ m 2 and an image size of 858 x 686 ⁇ m.
  • the CellSpotter® system acquires 4 rows of 35 images for each of the 4 filters resulting in 140 frames and 560 images per chamber.
  • the CellSpotter® acquisition program automatically determines the region over which the images are to be acquired, the number of images to acquire, the position of each image and the microscope focus to use at each position.
  • the image acquisition region is determined by moving the X and Y stages to 5 positions that should have an edge of the chamber visible in the image.
  • the software determines the edge location, draws a line on the image display where it has found the line, and gives the operator the ability to approve or override the selected edge location.
  • Two measurements are made on one of the long edges of the chamber to also determine the angular offset of the chamber relative to the X-axis of the stage. All images need to be in focus over the whole imaged area.
  • the depth of focus of the microscope is less than 10 ⁇ m. While the chamber surface is planar to within 10 ⁇ m, mechanical tolerances within the microscope stage, magnetic yoke and chamber may cause an angular skew in the Z-axis.
  • Figures 3A-3D shows the images of DAPI (Panel 3A) , DiOCl ⁇ (Panel 3B) , CK-PE (Panel 3C) and CD45-APC (Panel 3D) of one of the 140 frames obtained after processing a 7.5 ml blood sample from a patient with breast cancer.
  • DAPI DAPI
  • DiOCl ⁇ Panel 3B
  • CK-PE Pulsel 3C
  • CD45-APC Pulsel 3D
  • control image stained in the CK-PE image One of the boxes shows two nuclei and bright CK-PE staining corresponding to two cells.
  • the CD45-APC image showed no staining in the box confirming that the box contained two cells of epithelial cell origin.
  • the other box showed dim CK-PE staining and bright CD45-APC staining excluding this event as an epithelial cell.
  • An algorithm is applied on all of the images acquired from a sample to search for locations that stain for DAPI, DiOCl ⁇ and CK-PE. If the staining area is consistent with that of a control cell (DiOCl ⁇ , CK- , PE+) , the software assigns this location (box) to a control cell.
  • the data analysis software tabulates the number of control cells found in a sample. If the staining area is consistent with that of a potential tumor cell (DAPI+, DiOCl ⁇ -, CK-PE+) , the software stores the location of these areas in the database. The software displays thumbnails of each of the boxes for each of the parameters in rows. From left to right these thumbnails represent the nuclear (DAPI), cytoplasmic cytokeratin (CK-PE) , control cell (DiOCl ⁇ ) and surface CD45 (CD45-APC) staining. The composite images shown at the left show a false color overlay of the nuclear (DAPI) and cytoplasmic (CK-PE) staining.
  • the cell in row 201 is relatively small, a cluster of 3 tumor cells is present in row 202 and one very large cell is shown in row 204.
  • a control cell is shown in the top of the box. The area is displayed because of the CK-PE positive event below the control cell. The nuclei shown, however, belong to leukocytes and do not coincide with the CK-PE staining.
  • Row 205 shows debris that stains positively in all four filters and in row 206, the CK-PE positive event does not coincide with the DAPI staining.
  • Spiking 0 and 10 tumor cells into 7.5 ml aliquots of blood from 10 donors was done to test the sensitivity of the system.
  • the average control cell recovery in the 20 experiments was 85% with a coefficient of variation of 7.7%.
  • the certainty of the actual spike numbers decreases with the number of cells spiked.
  • the experiments were performed on two different days: one day the coefficient of variation in spiking 10 cells was 25% and on day two 15%. In the unspiked samples no tumor cells were found after processing.
  • tumor cells were detected in all spiked blood samples ranging from 6 to 15 tumor cells (mean 10.5 cells CV 32%).
  • the data clearly demonstrate that the sensitivity of the system is limited only by the blood volume processed.
  • six systems were placed at different sites. Blood samples from 99 healthy donors were processed in duplicate at these sites. The average recovery of the control cells across the six sites was 77.1 % with a coefficient of variation of 9.7%. As expected, the reproducibility between duplicate samples is better with a coefficient of variation of 4.9%.
  • the number of events that were classified by the software as potential tumor cell candidates varied between 10 and 304 events with a mean of 55.
  • Circulating tumor cells can be detected in the blood of patients with carcinomas, albeit at extremely low frequencies.
  • a system that can accurately and reliably enumerate and characterize CTC is needed to perform controlled clinical studies.
  • the number of CTC may represent tumor burden and changes in the CTC numbers could offer a means to evaluate the effectiveness of a given treatment.
  • Analysis of the CTC for the presence or absence of therapeutic targets could be used to guide treatment.
  • Detection of CTC in purportedly healthy individuals represents an advance in the early detection of cancer. If such early detection is possible a non-invasive, "whole body" biopsy of a solid tumor can be performed by a blood test.
  • the system produces a 320 ⁇ l liquid sample that is transferred to an analysis chamber and a magnetic device that causes all magnetically labeled cells in the sample to be pulled to the upper inside surface of the chamber for analysis.
  • Four-color fluorescent analysis is performed on the sample by the CellSpotter® system that enumerates internal control cells and identifies objects that potentially classify as tumor cells by their positive staining of the nucleus, cytoplasmic cytokeratin and their lack of cell surface staining for CD45. Thumbnails of all objects that potentially classify as tumor cells are presented in the user interface from which the user can make the ultimate judgment.
  • Sample preparation performed by the system provides advantages when compared to the manual preparation of blood samples as demonstrated by a higher recovery and better reproducibility of enumerated tumor cells.
  • Data from spiking experiments demonstrated an excellent linearity and sensitivity of the system.
  • duplicate blood samples from 99 normal donors were processed at six different sites.
  • Data across all sites demonstrated a level of reproducibility as assessed by recovery of internal control cells.
  • the average recovery of the internal control was 77.1% with a coefficient of variation that varied between 3.2% and 11.8% (mean 9.7%).
  • the sensitivity of the system was determined by the ability to detect CTC in patient samples and the specificity by identification of CTC in blood of normal donors.
  • 10 and 304 (mean 55) candidate CTC were found and 0 - 59 (mean 7) classified as CTC.
  • 13 events were classified as CTC by the operator, but review of the data revealed that this could be attributed to internal control cells that were weakly stained with the DiOC16.
  • EpCAM epithelial cell adhesion molecule
  • 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)).
  • the GA73.3 or MJ37 EpCAM antibodies recognizing two different epitopes on EpCAM (kindly provided by D Herlyn (Herlyn D, et al. J Immunol Methods. 73:157-167 (1984)) Wistar
  • An exemplary method for determining the tissue source of circulating epithelial cells employs cytochemical and immunological identification techniques.
  • the samples were incubated for 20 minutes at room temperature, washed twice in PBS for 5 minutes and then exposed to secondary rabbit anti-mouse Ig (Z0259, Dako Corp., Carpenteria, CA) for another 20 minutes. After two more washes, the samples were incubated with alkaline-phosphatase-anti-alkaline phosphatase (APAAP) rabbit Ig complexes for 15 minutes. Finally, the enzyme- substrate (New Fuchsin, Dako Corp. CA) was added resulting in the development of red precipitates. The nucleus was counterstained with hemotoxylin. The data were recorded using a Kodak digital camera attached to a light microscope. Data could be stored on CD for later reference . Sample analysis.
  • FIGs 5A and 5B The results obtained when tumor cells spiked into whole blood are isolated using the assay methods of the present invention are shown in Figures 5A and 5B.
  • Panel 5A shows analysis by microscopy and panel 5B shows analysis results obtained using flowcytometry .
  • Figs. 6A- 6C show three examples of the flowcytometric analysis of 10 ml blood samples obtained from one patient with metastatic breast carcinoma at three time points, and includes the correlative display of the anti-leukocyte versus anti-epithelial cell antibodies of the flowcytometric analysis.
  • Fig. 6 Panel A, 14 events are detected and are present in the location typical for epithelial cells.
  • Panel 6B 108 epithelial cells are detected and in Panel 6C, 1036 epithelial cells are detected.
  • Fig. 7 The dynamics of epithelial cell counts in the blood of 8 patients with metastatic disease are presented in Fig. 7.
  • the shaded area in the plots indicates the range at which positive events were detected in the controls. The plots also indicate when chemotherapy was administered.
  • Figure 7, panel A shows a patient with life threatening disease and 200 epithelial cells / 10 ml of blood at the time she entered the study. High dose adriamycine reduced the number within the normal range, but it rose again after adriamycine was discontinued. After a second course of adriamycine, the number of epithelial cells dropped significantly, but was still above the normal range.
  • Panel B shows the course of one patient over a period of 43 weeks.
  • the large size beads need to be removed from the cell surface prior to visualization or analysis.
  • the efficiency of cell selection with larger beads can be improved by increasing the concentration of beads and increasing the incubation time with continuous mixing to facilitate binding to rare target cells.
  • Dynal anti-epithelial cell beads (Dynal, NY) were used to test the efficiency of tumor cell selection from blood in a model study under optimum 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) were spiked into normal blood to determine the recovery after selection with beads. The tumor cells were pre- labeled with a fluorescent dye to differentiate them from blood cells during detection. The protocol was followed as recommended by the manufacturer.
  • SKBR-3 breast cancer cell line
  • SKBR-3 cells were pre-stained with a nucleic acid staining dye (Hoechst 33342) to allow detection after the selection by beads.
  • the blood was diluted with 5ml of Dulbecco ' s PBS containing 5mM EDTA and mixed with the diluted blood for 15 minutes at 4°C on a rocker.
  • lOO ⁇ l of Dynal anti-epithelial cell beads containing 50 x 10 6 beads were added to the blood sample and incubated for 30 minutes at 4°C with mixing on a rocker.
  • the number of beads used were similar to total white blood cells i.e. one bead per white cell.
  • the magnetically labeled cells were separated by placing the sample tube into Dynal MPC magnetic separator for 6 minutes . After aspirating the supernatant, the collected cells were resuspended in 3ml of Dulbecco ' s PBS containing 0.1% BSA. The sample tube was placed back into Dynal ' s MPC for 6 minutes to remove any carry-over blood cells. The magnetically bound cells were resuspended in 200 ⁇ l of Dulbecco' s PBS containing 0.1% BSA after aspiration of the supernatant.
  • the final sample containing selected tumor cells, non-specifically bound blood cells and excess free magnetic beads, was spotted onto an immunofluorescent slide to detect tumor cells.
  • the 200 ⁇ l sample was spotted into 10 different wells to disperse free magnetic beads.
  • the fluorescently stained tumor cells present in each well were counted using a fluorescent microscope. The results are shown in the Table V:
  • the final sample (200 ⁇ l) contained 50 xlO 6 beads in addition to selected tumor cells (10-17) and non-specifically bound leukocytes. The size of the beads (2.8 ⁇ m) is similar to that of certain blood cells and occupied most of the surface area on the slide. Therefore, to obtain recovery data, the sample had to be spotted onto several wells in order to sufficiently disperse free magnetic beads so as to allow for detection of recovered tumor cells.
  • tumor cells were pre-stained with a fluorescent nucleic acid dye and further staining was not necessary for detection.
  • tissue of origin of the magnetic bead-bound cells is often desirable. Such identification is performed using labeled antibodies to detect and characterize tumor cells present in clinical samples.
  • beads have to be removed from cell surfaces and separated from the sample following target cell selection, i.e. before analysis. This is not the case with magnetic nanoparticles because their size does not interfere with cell analysis.
  • this example shows that large magnetic beads may also be utilized in the methods disclosed herein for the efficient isolation of circulating tumor cells.
  • One method is to displace antibody from the cell surface by adding an excess specific competing reagent in excess that has higher affinity for the involved antigen or antibody.
  • This type of mechanism is used to release beads from CD34 selected cells in clinical applications using a peptide (Baxter Isolex 300) .
  • the peptide competes with CD34 antigen for binding to antibody on beads and releases the antibody-bead complex from cells.
  • Another method employs a reversible chemical linker between beads and antibodies.
  • the chemical linker can be inserted during the conjugation of antibodies to magnetic beads.
  • the chemical link can be cleaved under appropriate conditions to release beads from antibodies.
  • One of the methods currently in use employs a nucleic acid linker to link antibodies to magnetic beads.
  • the nucleic acid linker is a polynucleotide and can be hydrolyzed specifically using DNAse enzyme. Following hydrolysis of the nucleotide bonds present in the nucleic acid linker, the beads are released from the antibodies that remain bound to cells. The released beads can be removed from cell suspension by magnetic separation. The cells that are freed from beads can be used for further analysis by microscopy or flow cytometry .
  • Table VI each of the patients is listed and sorted according to the TNM (tumor, node, and metastasis) stage at the time of surgery followed by the years after surgery. Table VI also shows whether or not the patient received treatment (either chemotherapy or hormonal therapy) during the period studied.
  • TNM tumor, node, and metastasis
  • Table VI also shows whether or not the patient received treatment (either chemotherapy or hormonal therapy) during the period studied.
  • epithelial cells were found in the blood at a higher frequency than that found in the control group. Circulating epithelial cells were also found in 9 of 31 patients with no evidence of distant metastasis.
  • Fig. 8 shows two cells staining positive for cytokeratin and obtained from a patient with no evidence of metastatic disease at the time the blood was drawn.
  • Panel 8B shows a cell from a patient with metastatic disease in the past but in complete remission.
  • Panels 8C and 8D two cells are shown isolated from the blood of patient 25 at time point 6. The cell shown in Panel 8C has features consistent with malignancy whereas the cell in Panel 8D has the appearance of a normal squamous epithelial cell.
  • Tx therapy
  • CT chemotherapy
  • H hormonal therapy
  • - no therapy
  • 1,2,3,4,5,6,7 subsequent time point at which the number of epithelial cells was determined in years
  • EXAMPLE 5 Enumeration of circulating epithelial cells in patients diagnosed with breast cancer before surgical intervention.
  • Table VII summarizes the results obtained following similar clinical trials in which 13 controls and 30 patients with breast cancer were assessed using the assay of the invention.
  • n 12 14 5 11 mean 1.5 15.9 47.7 122 . 5
  • Flowcytometry was used to analyze the positive events obtained from 20 ml of blood from control individuals and from women with breast carcinoma.
  • the numbers of epithelial cells in the blood of controls are statistically different by t test (P ⁇ 0.01) and by Kruskall-Wallis nonparametric analysis (P ⁇ 0.001) from each of the three groups of the breast cancer patients.
  • Fig. 9 Three patients with metastatic disease of the prostate were assessed for the presence of circulating epithelial cells in their blood following chemotherapeutic treatment. The results are presented in Fig. 9. The data reveal that an increase in circulating epithelial cells in the blood is correlatable with disease activity.
  • the CTC number was 5 or more per 7 mL .
  • the CTC size was assessed by forward light scatter and in samples with 5 or more CTCs, 77% ⁇ 15% of these cells were larger than 10 ⁇ m.
  • the CTC and PSA values of four of these patients are shown in Figures 10E-H.
  • the CTC count was 5 or more, and 51% ⁇ 17% of the CTCs were larger than 10 ⁇ m.
  • the increase in CTCs paralleled the increase in the PSA level .
  • the method of the present invention can quantify CTCs and was used to assess the CTC changes during HRPC progression.
  • In vitro PC3 cell spiking experiments demonstrated a strong linear correlation (P? - 0.99) and an excellent recovery rate (74% ⁇ 9%) .
  • the detection limit of 0.8 + 1.2 cell in 7.5mL of blood was determined by analyzing the blood of 22 normal male donors.
  • the average CTC size was smaller in men with more advanced disease. Chemotherapy altered the number but not the CTC size, as we did not find changes in the CTC size to suggest cellular degradation before, during, or after administration of chemotherapy.
  • CTC counts can be reproducibly measured in patients with HRPC.
  • the changes in CTC levels mirrored disease progression.
  • the pattern and velocity of the CTC and PSA rise are different, suggesting that CTCs provide prognostic information independent of PSA.
  • the characterization of the CTC genotype and phenotype can guide future treatment and elucidate mechanisms of chemosensitivity and resistance.
  • the assay method of the present invention may be used to advantage in the assessment of patients with a variety of different cancer types. To illustrate, the method was also used to assess circulating epithelial levels in patients with colon cancer. There are over
  • Table X and Fig. 13 depict data obtained when colon cancer patients with evidence of metastases were assessed for the presence and number of circulating epithelial cells.
  • Rational clinical development of anticancer agents is impeded by infrequent access to repeat tumor biopsies for in vivo pharmacodynamic evaluation.
  • the methods of the present invention overcome this limitation by permitting assessment of drug effect in circulating tumor cells.
  • An additional pilot study to evaluate the ability of the immunomagnetic separation and automated fluorescent microscopic system of the invention to isolate, enumerate, and characterize circulating epithelial cells from the peripheral blood of patients (pts.) with metastatic colorectal cancer was performed. Twenty patients with measurable metastatic disease were enrolled. Fifty ml of peripheral blood were obtained at initiation of therapy and at disease reevaluation timepoints (6-10 week intervals).
  • Additional phenotyping by flow cytometry for epidermal growth factor receptor and thymidylate synthase expression to evaluate suitability of this technology for in vivo phar acodynamic assessment can also be performed in accordance with the methods of the present invention.
  • This study has demonstrated the feasibility of isolating circulating tumor cells from the blood of patients with metastatic colorectal cancer.
  • the present invention encompasses methods for assessing alterations in circulating tumor cells relative to tumor cells present in situ in a tumor mass. Such alterations may include for example, gain or loss of tumor diathesis associated molecules. Alterations in genotype or phenotype may also be examined.
  • EXAMPLE 8 TISSUE SOURCE IDENTIFICATION OF ISOLATED EPITHELIAL CELLS All of the aforementioned studies in patients reveal that there is an excess of circulating epithelial cells in patients who have cancer, compared to normal individuals or patients without cancerous diseases, including benign tumors. It is essential, however, to prove that these excess circulating epithelial cells are, in fact, cancer cells. This was accomplished by performing an experiment in which immunomagneticallly purified epithelial cells from patients with or without cancer were cytospun onto a glass slide and treated with anti-mucin. In addition, normal epithelial cells that were obtained from foreskin and blood from normal individuals, both used as controls, were also cytospun.
  • the methods disclosed herein enable the detection of cancer cells in the blood of patients with early tumors. Indeed, in 25 of 27 patients who were clinically determined to have organ-confined disease (early stage cancer) , we detected the presence of cancer cells in the blood. This means that the assay should detect cancer cells much earlier in those solid tumors that are normally detected late (10 9 -10 10 tumor cells) . Moreover, the test should allow detection of breast, prostate and colon cancer earlier, perhaps before detection of a primary tumor by conventional means.
  • the organ-origin of tumor cells in the blood for prostate can be established by staining with anti-prostate specific membrane antigen (PMSA) , anti-PSA (prostate specific antigen) , or other antibodies specific to the prostate in male subjects. For breast carcinoma in female patients, staining with anti-mammoglobin, anti-progesterone receptor, anti- estrogen receptor and anti-milk fat globulin antigen I and II will indicate a breast origin of tumor.
  • PMSA anti-prostate specific membrane antigen
  • anti-PSA
  • Each of the carcinomas described in the table above express tissue specific antigens whose corresponding antibodies can be used to determine the organ-origin of the circulating tumor cells.
  • the blood test of the invention can also be used to detect cancer cells in patients previously treated successfully for cancer and now in long-term complete remission. Indeed circulating epithelial cells, i.e., dormant tumor cells, have been detected in patients treated five or more years previously and who appear to be clinically free of tumor. This explains why recurrence in patients can occur many years, even decades after apparently successful treatment. In fact, accumulating evidence suggests that the recurrence of breast cancer occurs at a slow steady rate approximately 10-12 years after mastectomy.
  • the present invention may be used to advantage to diagnose cancer in presently asymptomatic patients.
  • a patient with a two-year history of high PSA levels (>12 ⁇ g/ml)
  • had a needle biopsy of the prostate performed two weeks prior to the analysis set forth below.
  • the biopsy did not reveal the presence of malignancy.
  • a prior biopsy performed 18 months earlier was also negative.
  • the patient Before obtaining a 20 ml blood sample, the patient was given a digital rectal exam and a gentle massage of his enlarged prostate with the intention of increasing the occurrence of tumor cells in the blood.
  • the blood sample was enriched using the methods of the present invention.
  • the enriched fraction was examined by microscopy employing a Wrights-Giemsa stain. Morphological examination of the isolated cells revealed their malignant character. Clearly this patient had cancer. Given the high PSA levels observed, a diagnosis of prostate cancer is likely.
  • the origin of the cells may be determined using appropriate reagents as described herein. The results presented in this example reveal that the methods of the present invention can be used to detect cancers that might otherwise go undetected.
  • HER-2 c-erbB2
  • CTCs circulating tumor cells
  • HER-2 is exemplified herein, it is highly desirable that additional tumor-diathesis associated molecules on or in the tumor cells be identified and assessed in this manner. Suitable tumor diathesis associated molecules that may be assessed following isolation of circulating tumor cells are set forth in Table XI.
  • Exemplary approaches for detecting alterations in tumor diathesis associated molecules such as nucleic acids or polypeptides/proteins associated with malignancy include: a) comparing the sequence of predetermined nucleic acid in the sample with the corresponding wild-type nucleic acid sequence to determine whether the sample from the patient contains mutations; or b) determining the presence, in a sample from a patient, of tumor diathesis associated molecules polypeptide and, if present, determining whether the polypeptide is full length, and/or is mutated, and/or is expressed at the normal level; or c) using DNA restriction mapping to compare the restriction pattern produced when a restriction enzyme cuts a sample of tumor diathesis associated molecules nucleic acid from the patient with the restriction pattern obtained from the cognate normal gene or from known mutations thereof; or, d) using a specific binding member capable of binding to a tumor diathesis associated molecules nucleic acid sequence (either normal sequence or known mutated sequence) , the specific binding member comprising nucleic acid hybridizable with the sequence
  • Alterations in protein molecules e.g., those arising from deletion or point mutation in the encoding nucleic acids may be assessed using conventional methods which are well known to those of ordinary skill in the art. Such methods include gel electrophoresis, western blotting, HPLC, and FPLC. Alterations in nucleic acid molecules which are associated with malignancy may also be assessed using conventional methods.
  • tumor diathesis associated molecules may include receptors for HER-2, estrogen, and progesterone. Problems arise as the disease progresses, since changes in the phenotype of the tumor cells often occur after the original diagnosis, and resistance to a treatment can only be inferred after the treatment has failed. Assessing the presence of target tumor diathesis associated molecules on CTCs before and during treatment constitutes a real-time, "whole body" biopsy of the tumor.
  • Tissue blocks from the patient's primary tumors were collected and slides were prepared from paraffin-embedded tissue sections.
  • the slides were evaluated for HER-2 expression using the HercepTest® (DAKO, Carpinteria, CA ) according to the manufacturer's instructions. Positive and negative slides were reviewed by a single pathologist and scored according to the manufacturer's guidelines using a scale from 0 to 3+.
  • desthiobiotin was coupled to EpCAM-labeled magnetic nanoparticles to form CA-EpCAM as described in the previous examples.
  • CA-EpCAM ferrofluid and a buffer containing streptavidin were then added to the sample to achieve this increase in the magnetic labeling of the cells.
  • Desthiobiotin on the CA-EpCAM ferrofluid was subsequently displaced by biotin, which is contained in the permeabilization buffer described below, thereby reversing the cross linking between the CA-EpCAM ferrofluids .
  • the sample was immediately placed in a magnetic separator composed of four opposing magnets for 10 minutes (QMS17, Immunicon, Huntingdon Valley, PA). After 10 minutes, the tube was removed from the separator, inverted 5 times, and returned to the magnetic separator for an additional 10 minutes. This step was repeated once more and the tubes were returned to the separator for 20 minutes. After separation, the supernatant was aspirated and discarded. The tube was removed from the magnetic separator and the fraction collected on the walls of the vessel was resuspended with 3ml of BSA containing PBS. The suspension was placed in the magnetic separator for 10 minutes and the supernatant was aspirated and discarded.
  • the cells were resuspended in 200 ⁇ l of a biotin containing permeabilization buffer (Immuniperm, Immunicon Corp.) to which Mab-fluorochrome conjugates were added at saturating conditions.
  • the Mabs consisted of a Phycoerythrin (PE) conjugated anti- cytokeratin Mab Cll recognizing keratins 4,6,8,10,13, and 18, (Immunicon Corp.), Peridinin Chlorophyll Protein (PerCP) -labeled anti-CD45 (Hle-1, BDIS, San Jose, CA) and cyanin 5 (CY5) -labeled anti-HER-2.
  • PE Phycoerythrin
  • PerCP Peridinin Chlorophyll Protein
  • the MAb anti-HER-2 designated HER-81, recognizes an epitope on the extracellular domain of HER-2 and does not cross block with trastuzumab or its murine parent 4D5. It is a murine IgG ⁇ ⁇ with a Kd of 10 "10 M on BT474 breast carcinoma cells. After incubating the cells with the Mabs for 15 minutes, 2 ml of cell buffer (PBS, 1%BSA, 50mM EDTA) was added and the cell suspension was magnetically separated for 10 minutes. After discarding the non-separated suspension, the collected cells were resuspended in 0.5 ml of PBS to which the nucleic acid dye used in the
  • Procount system was added (Procount, BDIS, San Jose CA) .
  • 10,000 fluorescent counting beads were added to the suspension to verify the analyzed sample volume (Flow-Set Fluorospheres, Coulter, Miami, FLA) .
  • Cells of the prostate cancer cell line PC-3 and the breast cancer line SKBR-3 were cultured in flasks containing 10 ml RPMI-1640 supplemented with 10%FCS. Cells were harvested from the flasks after trypsin treatment, washed and resuspended to obtain the desired cell concentration. For quantitative assessment of HER-2 density on both cell lines, 20 , 000 cells were stained with the Mab HER-81 conjugated to PE (HER-2 PE) . For calibration of expression levels of HER-2 100 ⁇ l of cell suspension containing approximately 3,000 cells was spiked into 5 ml of blood.
  • Sample analysis Samples were analyzed on a FACSCalibur flow cytometer equipped with a 488nm Argon ion laser and a 635nm laser diode (BDIS, San Jose, CA) .
  • Data acquisition was performed with CellQuest (BDIS, San Jose, CA) using a threshold on the fluorescence of the nucleic acid dye. The acquisition was halted after 8000 beads or 80% of the sample was analyzed.
  • Multiparameter data analysis was performed on the listmode data (Paint-A-Gate Pro , BDIS, San Jose, CA) .
  • Analysis criteria included size defined by forward light scatter, granularity defined by orthogonal light scatter, positive staining with the PE-labeled anti-cytokeratin MAb and no staining with the PerCP- labeled anti-CD45 Mab.
  • the flow cytometer was calibrated to assess the density of HER-2 on cells using phycoerythrin (PE) -labeled beads with known numbers of fluorochrome molecules (QuantiBRITE PE, BDIS, San Jose, CA) .
  • the densities of HER-2 on the breast cancer cell line, SKBR-3, and prostate cancer cell line, PC-3 were then assessed by measuring the fluorescence intensity of cells treated with PE-anti-HER- 2 under saturating conditions.
  • HER-2 receptors on SKBR-3 and PC-3 cells were 850,000 and 9,500, respectively.
  • Enumeration of CTCs To obtain the sensitivity needed for the enumeration of CTCs, a combination of technologies was required. First, an enriched sample of EpCAM + cells in the blood was prepared by mixing the cells with colloidal paramagnetic particles coated with MAbs specific for EpCAM, followed by magnetic separation. This immunomagnetic sample enrichment is performed to reduce both the sample volume and the number of background hematopoietic cells.
  • the remaining cells were then labeled with PE-anti-cytokeratin to identify epithelial cells, PerCP-anti-CD45 to identify leukocytes, Cy5-anti HER-2 to determine the expression of HER-2, and a nucleic acid dye was added to exclude residual erythrocytes, platelets and other non-nucleated "events”. Magnetic separation (i.e., washes) was used throughout the sample preparation to eliminate excess labeling reagents, to reduce carryover of non-target cells, and to permit the resuspension of cells in the desired volume. The samples were analyzed by flowcytometry with each event being characterized by two light scatter and four fluorescence parameters.
  • Figure 15 is an example of the flowcytometric analysis of a 5 ml blood sample obtained from a patient with metastatic breast carcinoma.
  • Panel 15A shows the correlative display of PerCP-anti-CD45 versus PE-anti-Cytokeratin;
  • Panel 15B shows the PE-anti- cytokeratin versus Nucleic Acid Dye;
  • Panel 15C shows the forward and right angle scatter, and
  • Panel 15D shows the Cy5- anti-HER-2 versus PE-anti-Cytokeratin.
  • the location of beads and leukocytes are indicated in the Panels and are depicted in black.
  • the gates drawn in Panels 15A, 15B and 15C indicate the regions typical for CTCs (CD45 " , Cytokeratin + , >4 ⁇ m, Nucleic acid + ) .
  • CTCs highlighted black small squares
  • CTCs were considered to be HER-2 + if they exceeded the background staining of leukocytes as indicated by the dotted line in Panel 15D.
  • the same gates shown in the figure were used to analyze all the samples in this study. In 5 ml of blood from 22 controls 1.5 + 2.1 events per subject were detected in the regions that classified as CTCs. None of these events were associated with expression of HER-2. In contrast 5-214 CTCs/5 ml were detected in 10 of 19 blood samples from patients with measurable disease who were starting an initial or new line of therapy.
  • HER-2 on CTCs from three breast cancer patients is shown in Figure 16B, 16C and 16D.
  • Table XI shows the treatment modality, response to therapy, HER-2 expression on primary tissue, months between the tissue biopsy and the assessment of CTC, baseline and post-treatment CTCs and expression of HER-2 on CTCs from 19 breast cancer patients. Tissue blocks of the primary tumor obtained at the time of diagnosis were assayed for HER-2 expression. The time between diagnosis (tumor biopsy) and CTC determination varied greatly (Table XI) . The tissue blocks of seven of the 19 patients were positive for HER-2 (2+ or 3+) by Herceptest staining. In 6/7 patients, CTCs were detected and in all 6, the CTCs expressed HER-2, (Table XII) . In one patient, CTCs expressed HER-2 whereas the tissue section did not. The percentage of CTCs that expressed HER-2 and the density of the HER-2 on the surface of the CTCs varied considerably.
  • Pt# patient number.
  • HER-2 expression the percentage of CTCs expressing H ⁇ R-2. In the subsequent figures the actual number of CTCs at the four different densities is shown at the different time points.
  • the number of CTCs decreased from 214 to 79 four weeks after initiation of treatment and only 14% expressed HER-2.
  • the patient's clinical status was based on CT and bone scans taken after the course of treatment although a clear reduction in CTCs was observed.
  • trastuzumab Herceptin®
  • Herceptin® trastuzumab
  • HER-2-overexpressing breast cancer has added an important regimen to the therapies available for patients with this disease (Baselga et al . Proc. Am. Soc. Clin. One. (1995) 14:103a; Pegra et al . J. Clin. Oncol. (1998) 16:2659-71; Cobleigh et al . J. Clin. Oncol. (1999) 17:2639).
  • the development and utilization of such target-directed therapies requires a 'real time' accurate, sensitive, specific and reliable in-vitro diagnostic assay.
  • the assay must be capable of detecting not only the patient subpopulation likely to benefit from a given therapy but the patient subset in whom resistance to that treatment has developed.
  • Candidacy for trastuzumab therapy currently requires a positive diagnosis by either by immunohistochemistry (positive HER-2 staining of the tumor) or evidence of amplification of the HER-2 gene as determined by fluorescence in-situ hybridization (FISH) .
  • FISH fluorescence in-situ hybridization
  • CTCs measured at several time points during the day did not change, whereas substantial increases were found over a longer period of time during which the disease progressed.
  • CTCs were detected in 5 ml of blood from 10 of 19 patients with stage III and IV breast cancer.
  • larger blood volumes and automation of the sample preparation procedure can be used to increase the sensitivity of the assay.
  • CTCs from patients with HER-2-overexpressing tumors were HER- 2 + supports the rationale of the assay. More important was the fact that in three patients, a phenotypic conversion from HER-2 " to HER-2 + CTCs was observed.
  • HER-2 overexpression was not detected on the CTCs prior to initiation of a new line of treatment but was detected on CTCs that underwent a concomitant substantial increase in number during the course of treatment.
  • the conversion to HER-2 + CTCs might signify conversion to a more aggressive phenotype.
  • the observation that expression of HER-2 was inversely related to expression of cytokeratin, a cytoskeletal protein associated with cellular differentiation supports this hypothesis (Schaafsma et al . in Rosen, P.P., Fechner, R.E., eds. Pathology Annual vol. 29 (1994) pp. 21-62)
  • the ability to detect changes in HER-2 expression is of clinical importance in regards to selection of HER-2 targeted therapy and chemotherapy.
  • HER-2 is exemplified herein, alterations in many other tumor diathesis associated molecules and markers can occur as a tumor cell becomes more malignant. Molecules that exhibit alterations associated with malignancy include without limitation, mdr, thymidylate synthase, FSFR, p53, ras oncogenes, CD 146, src, MUC1, uPA, PAI-1, ACT and many others.
  • Table XII provides a list of biological molecules that may be assessed by the clinician, provide a more accurate diagnosis of the patient's condition and, more importantly, to devise the appropriate therapeutic regimen for treatment. For example, pS2/pNR-2 + breast cancer indicates that the patient is likely to respond to endocrine therapy. Additional tumor diathesis associated molecules which are often altered during malignant progression and which may be analyzes in isolated circulating tumor cells are listed below. This list is meant to be illustrative only, and not limited to the molecules set forth. TABLE XII Theraputic Targets
  • NSE Laminin Receptor Neuron Specific Enolase
  • GST Glutathion S Transferase
  • kits comprising the reagents used to perform the assay of the invention.
  • Such kits are designed for particular applications.
  • Reagents may be assembled to facilitate screening of patients for circulating rare cells, including but not limited to tumor cells.
  • the kits contain colloidal magnetic particles comprising a magnetic core material, a protein base coating material and a biospecific ligand which binds specifically to a characteristic determinant present on the cancer cell to be isolated.
  • the kit also includes at least one additional biospecific reagent that has affinity for a second characteristic determinant on the cancer cell to be isolated which differs from the determinant recognized by the biospecific ligand.
  • the kit also includes a cell specific dye for excluding non-nucleated cells and other non- target sample components from analysis.
  • An exemplary kit also comprises reagents for detecting at least one tumor diathesis associated molecule.
  • Also provided in the kit is a Cell Spotter or Cell Tracks cartridge as described in Example 2.
  • a typical kit according to this invention may include anti -EpCAM coupled directly or indirectly to magnetic nanoparticles, and a pair of monoclonal antibodies, the first antibody recognizing a cancer specific determinant and the second antibody having affinity for a non-tumor cell determinant, e.g., a pan leukocyte antigen.
  • Reagents which also detect at least one tumor diathesis associated molecule are also provided in the kit.
  • the kit also contains a nucleic acid dye to exclude non-nucleated cells from analysis.
  • the kit of the invention may optionally contain a biological buffer, a permeabilization buffer, a protocol, separation vessels, analysis chamber, positive cells or appropriate beads and an information sheet.
  • kits described above may also be produced to facilitate diagnosis and characterization of particular cancer cells detected in circulation.
  • the kits contain all of the items recited above, yet also preferably contain a panel of cancer specific monoclonal antibodies.
  • a kit for diagnosis may contain anti-MUC-1, anti-estrogen, anti-progesterone receptor antibodies, anti-CA27.29, anti-CAl5.3, anti-cathepsin D, anti-p53, anti-urokinase type plasminogen activator, anti-epidermal growth factor, anti-epidermal growth factor receptor, anti-BRCAl, anti-BRCA2, anti-prostate specific antigen, anti-plasminogen activator inhibitor, anti-Her2-neu antibodies or a subset of the above.
  • kits are also provided for monitoring a patient for recurring disease and/or residual cells following eradication of the tumor.
  • the type of cancer will already have been diagnosed.
  • the kit will contain all of the reagents utilized for screening biological samples for cancer yet also contain an additional antibody specific for the type of cancer previously diagnosed in the patient.
  • the kit might contain anti-MUC-1.
  • the kit may contain anti-Her2-neu.
  • kits of the invention may be customized for screening, diagnosing or monitoring a variety of different cancer types.
  • the antibodies or complementary nucleic acids included in the kit would be specific for target molecules present in prostate tissue. Suitable antibodies or markers for this purpose include anti-prostate specific antigen, free PSA, prostatic acid phosphatase, creatine kinase, thymosin b-15, p53, HPC1 basic prostate gene and prostate specific membrane antigen.
  • an antibody specific for carcinoembryonic antigen CEA
  • CEA carcinoembryonic antigen
  • Kits utilized for screening patients with bladder cancer may contain antibodies to nuclear matrix protein (NMP22) , Bard Bladder tumor antigen (BTA) or fibrin degradation products (FDP) . Markers and tumor diathesis associated molecules are known for many different cancer types .
  • NMP22 nuclear matrix protein
  • BTA Bard Bladder tumor antigen
  • FDP fibrin degradation products
  • kits of the invention may be further studied for morphology, RNA associated with the organ of origin, surface and intracellular proteins, especially those associated with malignancy. Based on existing information on such molecules, it should be possible to determine from their expression on the isolated cell, the metastatic potential of the tumor via analysis of the circulating cells. It is an object of the invention to provide kits for any cancer for which specific markers are known. A list summarizing tumor diathesis associated molecules and the usefulness and/or indication follows:
  • the following table provides different cytokeratin markers that may be used to assess tissue origin of cells isolated using the methods of the present invention.
  • the kit starts with reagents, devices and methodology for enriching tumor cells from whole blood.
  • An exemplary kit for detecting breast cancer cells would contain that will assess six factors or indicators.
  • the analytical platform needs to be configured such that the reporter molecules DAPi, CY2 , CY3, CY3.5, CY5, and CY5.5 will be discriminated by the appropriate excitation and emission filters.
  • the analytical platform in this example uses a fluorescent microscope equipped with a mercury arc lamp, and the appropriate filter sets for assessing the wavelengths of the detection labels employed. All of the markers are introduced at one time with this method. DAPi, which is excited with UV light, stains nucleic acids, and will be used to determine the nuclear morphology of the cell.
  • CAM 5.2 labeled with CY2 will be used to stain the control cells.
  • CY3 labeled ⁇ -cytokeratin will be used to label cytokeratins 7, 8, 18, and 19.
  • 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 estrogen receptors.
  • Examples of different types of cancer that may be detected using the compositions, methods and kits of the present invention include apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell and transitional cell reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcom
  • the present invention is not limited to the detection of circulating epithelial cells only. Endothelial cells have been observed in the blood of patients having a myocardial infarction. Endothelial cells, myocardial cells, and virally infected cells, like epithelial cells, have cell type specific determinants recognized by available monoclonal antibodies. Accordingly, the methods and the kits of the invention may be adapted to detect such circulating endothelial cells. Additionally, the invention allows for the detection of bacterial cell load in the peripheral blood of patients with infectious disease, who may also be assessed using the compositions, methods and kits of the invention.

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Abstract

L'invention concerne des méthodes et des compositions destinées à détecter des cellules tumorales circulantes et à analyser lesdites cellules en vue de déceler des altérations dans les molécules associées à la diathèse tumorale.
PCT/US2002/005233 2001-02-16 2002-02-19 Methodes et reactifs destines a l'isolement rapide et efficace de cellules cancereuses circulantes WO2003065042A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
JP2003564585A JP2005516217A (ja) 2001-02-16 2002-02-19 循環している癌細胞の急速かつ効果的な単離のための方法および試薬
BR0207290-4A BR0207290A (pt) 2001-02-16 2002-02-19 Método para analisar um paciente quanto à presença de uma malignidade, célula maligna isolada das células cultivadas, vacina de tumor, molécula associada com diátese de tumor alterada, métodos para determinar alterações em moléculas associadas com diátese de tumor como um meio para predizer a eficácia da terapia, como um meio para analisar dosagem apropriada para terapia, como um meio para monitorar eficácia da terapia, como um meio para analisar progressão de câncer, para realizar uma biópsia de corpo inteiro em um paciente, e para identificar alterações em uma célula de tumor circulante relativamente a células presentes numa massa de tumor in situ, e, kit de teste para analisar uma amostra de paciente quanto à presença de uma célula maligna não-hematopoiética
KR10-2003-7010790A KR20030080002A (ko) 2001-02-16 2002-02-19 순환 암 세포를 신속하고 효과적으로 분리하기 위한 방법및 시약
AU2002306561A AU2002306561A2 (en) 2001-02-16 2002-02-19 Methods and reagents for the rapid and efficient isolation of circulating cancer cells
CN02807927.2A CN1871517A (zh) 2002-02-19 2002-02-19 快速有效分离循环癌细胞的方法和试剂
IL15725602A IL157256A0 (en) 2001-02-16 2002-02-19 Methods and reagents for the rapid and efficient isolation of circulating cancer cells
EP02806645A EP1360496A4 (fr) 2001-02-16 2002-02-19 Methodes et reactifs destines a l'isolement rapide et efficace de cellules cancereuses circulantes
CA002438112A CA2438112A1 (fr) 2001-02-16 2002-02-19 Methodes et reactifs destines a l'isolement rapide et efficace de cellules cancereuses circulantes

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US26927101P 2001-02-20 2001-02-20
US26927001P 2001-02-20 2001-02-20
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CA (1) CA2438112A1 (fr)
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US8969021B2 (en) 2001-10-11 2015-03-03 Aviva Biosciences Corporation Methods and compositions for detecting non-hematopoietic cells from a blood sample
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US10329353B2 (en) 2013-03-05 2019-06-25 Board Of Regents, The University Of Texas System Specific detection tool for mesenchymal and epithelial-mesenchymal transformed circulating tumor cells
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EP1360496A4 (fr) 2005-03-09
BR0207290A (pt) 2005-06-07
AU2008249153A1 (en) 2008-12-11
KR20030080002A (ko) 2003-10-10
CA2438112A1 (fr) 2003-08-07
AU2008249153B2 (en) 2012-05-10
IL157256A0 (en) 2004-02-19
EP1360496A1 (fr) 2003-11-12
AU2002306561A2 (en) 2003-09-02
JP2005516217A (ja) 2005-06-02

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