WO1999044064A1 - Methode et compositions de detection differentielle de cellules tumorales et de cellules metastatiques - Google Patents

Methode et compositions de detection differentielle de cellules tumorales et de cellules metastatiques Download PDF

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Publication number
WO1999044064A1
WO1999044064A1 PCT/US1999/004005 US9904005W WO9944064A1 WO 1999044064 A1 WO1999044064 A1 WO 1999044064A1 US 9904005 W US9904005 W US 9904005W WO 9944064 A1 WO9944064 A1 WO 9944064A1
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Prior art keywords
cells
sample
molecule
detector
cell
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PCT/US1999/004005
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English (en)
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Emilio Barbera-Guillem
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Cli Oncology, Inc.
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Priority to CA002322282A priority Critical patent/CA2322282A1/fr
Priority to AU27856/99A priority patent/AU2785699A/en
Publication of WO1999044064A1 publication Critical patent/WO1999044064A1/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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/55—IL-2

Definitions

  • the present invention relates to methods for determining if a specific subpopulation of cells is present in a sample comprising a complex biological fluid. More particularly, the present invention is directed to a flow cytometric method for the differential detection and enumeration of metastatic cells in a sample comprising a complex biological fluid that may contain an admixture of (various populations of normal and/or malignant) cells.
  • Metastasis is the spread of malignant tumors to secondary sites remote from the original or primary tumor. Metastasis presents a cancer clinician with difficulty in diagnosing and treating the malignant tumor because (a) metastases may be comprised of as little as one or a few cells thereby evading clinical diagnosis even with modern techniques; (b) often metastases have already been seeded by the time a patient is diagnosed with a malignant non- lymphoid tumor (Silverberg et al . , 1989, CA Cancer J. Clin .
  • a rapid, simple and efficient method of screening for subsequent recurrence/regrowth of tumor can be a cost- effective alternative to the relatively high cost, inconvenience, and lack of sensitivity of detection of micro- metastases, of post-therapeutic evaluations for residual or metastatic disease using radiographic techniques or imaging techniques (CT scans, NMR scans).
  • CT scans, NMR scans radiographic techniques or imaging techniques
  • Measurement of tumor cell-associated (either internal or membrane bound) IL-2Ra in experimental tumors and human tumors shows a correlation between tumor cell-associated expression of IL- 2Ra and the metastatic potential of a primary tumor, i.e. the likelihood that the primary tumor has already, or will, metastasize. While such diagnostic and prognostic methods are useful, they require a tumor specimen to measure the tumor cell-associated IL-2Ra. In that regard, problems may occur in examining tumor tissue as a specimen.
  • the primary tumor is inaccessible, or performing a biopsy of primary tumor is clinically very dangerous.
  • IL-2Ra also known as p55
  • tissue samples are obtained in a biopsy, thereby raising the issue of tumor sampling; i.e., the representativeness of the biopsy sample relative to the whole tumor.
  • the ability to detect micrometastases in complex biological fluids provides a more accurate view of metastasis than does the detection of primary tumor cells having a high potential to metastasize. Detection of metastatic cells in complex biological fluids is further complicated by the expression of IL-2Ra by T lymphocytes that may normally be present in the complex biological fluid.
  • Such a method may aid in the early detection of micrometastases, whether it be micrometastases associated with tumor recurrence or regrowth, in a manner which can distinguish between metastatic cells, malignant primary tumor cells, and cells other than metastatic cells (e.g., normal blood cell populations), present in a complex biological fluid.
  • a primary object of the invention is to provide a method for the differential detection of metastases of solid, non-lymphoid tumors by measuring multiple parameters by flow cytometric analyses.
  • Another object of the invention is to provide a method for the differential detection and enumeration of metastases of solid, non-lymphoid tumors by measuring multiple parameters by flow cytometric analyses.
  • Another object of the invention is to provide a method for the differential detection and enumeration of metastases of solid, non-lymphoid tumors by measuring multiple parameters by flow cytometric analysis, and which can distinguish between metastatic cells, malignant primary tumor cells, and cells other than metastatic cells (e.g., normal blood cell populations), circulating in a complex biological fluid.
  • a further object of the invention is to provide a method for staging malignant disease in an individual by differentially detecting and enumerating, in a complex biological fluid from the individual, metastatic cells by flow cytometric analysis using multiple parameters.
  • a further object of the present invention is to provide a method for the differential detection of micro- metastases in a complex biological fluid, the detection of which may be an indication of tumor recurrency or tumor regrowth
  • FIG. 1 is a histogram showing forward scatter (FS) and side scatter (90°LS) used to gate nucleated events.
  • FIG. 2 is a histogram showing forward scatter (FS) and Ber-EP4 fluorescence (BER-EP4); and a new gate formed which represents Ber-EP4 positive cells ("R2") .
  • FIG. 3 is a histogram showing Ber-EP4+ gated cells (FS) and IL-2Ra+ cells (CD 25) .
  • FIG. 4 is a histogram illustrating the level of fluorescence background events ("Events") detected, in analysis 'of a negative control comprising a fluorescence- labeled IgG isotype matched antibody.
  • Cell molecule is a term used hereinafter for the purposes of the specification and claims to refer to a ligand for which a detector molecule has binding specificity, wherein the ligand is selected from the group consisting of IL-2Ra or an epithelial cell antigen. Depending on the method of preparing the sample for flow cytometric analysis, the cell molecule may either be a cell surface molecule, or an intracellular molecule.
  • Complex biological fluids or “body fluids” are terms used hereinafter for the purposes of the specification and claims to refer to body fluids of an individual in which metastatic cells and cells other than metastatic cells (normal and/or malignant) can circulate.
  • Such fluids include, but are not limited to blood; lymph; ascitic fluid; pleural fluid; peritoneal fluid; mucous fluids (e.g., bronchial, vaginal, or prostate); other effusions associated with solid non-lymphoid tumors; and tissue cell suspensions, wherein the tissue comprises an aspirate or biopsy of liver, lung, brain, lymph node, bone marrow, adrenal gland, breast, colon, pancreas, stomach, or reproductive tract.
  • tissue comprises an aspirate or biopsy of liver, lung, brain, lymph node, bone marrow, adrenal gland, breast, colon, pancreas, stomach, or reproductive tract.
  • detectable moiety is used herein, for purposes of the specification and claims, to mean a label molecule that is directly or indirectly detectable by flow cytometry, as known to those skilled in the art of flow cytometry.
  • the detectable moiety may be bound to a monoclonal antibody (mAb) , or polyclonal antibody, or aptamer, having binding specificity for the cell molecule to be detected, using covalent or noncovalent means to form a "detector molecule" for use in a flow cytometric method.
  • epithelial cell antigen is used herein, for purposes of the specification and claims, to mean a molecule found on the surface and/or in the cytoplasm of most (if not all) simple epithelial cells, but not detectable (within the limits of detection using an antibody and relative to nonspecific background) with mesenchymal tissue such as lymphoid tissue or lymphocytes.
  • Such an epithelial cell antigen may include, but is not limited to, keratin, cytokeratin, and the antigen ("Ber-EP4") recognized by mAb Ber-EP4.
  • mAbs to keratin or cytokeratin include LE61, CAM 5.2, CK2, IT-Ks20.8, and others.
  • an antibody with binding specificity for the epithelial cell antigen can be used with specificity to determine whether a cell reacted therewith is epithelial in nature or origin; i.e., if the cell is an epithelial cell or a tumor cell of epithelial origin or nature.
  • the epithelial cell antigen according to the present invention is epithelial cell-specific, and not tumor-specific (as distinguished from the tumor-specific antigens disclosed in U.S. Patent No. 5,536,642), as antibodies to epithelial cell antigen cannot distinguish between normal epithelial cells and tumor cells of epithelial origin.
  • epithelial tumor-specific antigens which are epithelial cell-specific
  • epithelial tumor-specific antigens which recognize a tumor-associated antigen not normally expressed, or not accessible (e.g., cryptic) to antibody, in normal (non-neoplastic) epithelial cells of the same tissue type.
  • mAb Ber-EP4 is directed against two glycopeptides present on the surface and in the cytoplasm of all human epithelial cells except the superficial layers of squamous epithelia, hepatocytes, and parietal cells; but does not react with mesenchymal tissue (Passlick et al., 1996, Eur . J. of Cancer, 32A: 141-5). In addition to immunoreacting with simple epithelia, mAb Ber-EP4 has been found to immunoreact with solid non-lymphoid tumors including metastatic cells of adenocarcinoma in serous effusions (De Angelis et al .
  • metalastases or “metastatic cell” or “micrometastases” are used herein, for purposes of the specification and claims, to mean cells which have metastasized, or in the process of metastasizing, from a primary tumor where- in the primary tumor is a solid, non-lymphoid tumor, as will be more apparent from the following embodiments.
  • Parameters is used herein, for purposes of the specification and claims, to mean indicia measurable by flow cytometers including, but not limited to, light scatter produced by a cell, electrical impedance produced by a cell, and an emission or signal produced by the detectable moiety portion of the detector molecule bound to a cell.
  • Light scatter is the light scattered by a cell as it passes through an incident beam of light directed at the sensing region of a flow cytometer.
  • the scattered light may be used to determine one or more cell properties, including shape, size (forward scatter), index of refraction, granularity (side scatter), and roughness. Changes in electrical impedance as each cell passes through the sensing region can be used to determine cell volume.
  • fluorescent molecules used as a detectable moiety
  • a wide latitude of choice can be exercised in selecting one or more suitable light sources for use in the method of the present invention.
  • multiple fluorescent analyses e.g., detecting and measuring more than one fluorescence emission from a cell having bound thereto 2 types of detector molecules with each detector molecule recognizing a different cell molecule than the other
  • a combination of filters may be used to permit simultaneous detection of the spectrally separated emissions of two different, excited fluorescent molecules to provide information on multiple detector molecule use in a single flow cytometric run (see, e.g., U.S. Patent No. 4,727,020).
  • differentially detected are primary tumor cells and/or micrometastases of solid non-lymphoid tumors in blood or other body fluids of an individual.
  • measured in a complex biological fluid are cells expressing either epithelial cell antigen (representing a primary tumor cell) or a combination of epithelial antigen and IL-2Ra (representing a metastatic cell) in conjunction with other parameters by flow cytometry.
  • the epithelial cell antigen is Ber- EP4.
  • the detection in complex biological fluid of cells expressing an epithelial cell antigen, or a combination of epithelial cell antigen and IL-2Ra can be used for aiding in the diagnosis of metastases (an accurate assessment of micrometastases present in a complex biological fluid) ; providing an accurate assessment of residual cancer; monitoring an individual in cancer remission for recurrence; early detection of cancer recurrences; and staging the malignant disease in a tumor bearing individual.
  • the method of the present invention is a method for discriminating between primary tumor cells (including primary tumor cells residual from anticancer treatment), metastatic cells, and nonmalignant cells that may be present in a sample of body fluid.
  • the method of the present invention comprises the steps of: obtaining a sample of body fluid from an individual; adding to the sample at least two detector molecules, wherein a first detector molecule has binding specificity for a cell molecule comprising IL-2Ra, and a second detector molecule has binding specificity for a cell molecule comprising an epithelial cell antigen, and the detectable moiety on the first detector molecule has a peak emission spectrum which is distinguishable from the peak emission spectrum of the detectable moiety on the second detector molecule; and analyzing cells present in the sample in a flow cytometer which is capable of detecting and distinguishing the emission spectra of the detector molecules, and light scattering for each cell present in the sample; wherein primary tumor cells (including residual tumor) present in the sample are detectable by expression of the epithelial cell antigen
  • kits for detecting primary tumor cells, metastatic cells, or a combination thereof, in a body fluid comprising a detector molecule having binding specificity for human IL-2Ra, a detector molecule having binding specificity for an epithelial cell antigen expressed by human cells, wherein the detectable moiety on the detector molecule having binding specificity for human IL-2Ra has a peak emission spectrum which is distinguishable from the peak emission spectrum of the detectable moiety on the detector molecule having binding specificity for an epithelial cell antigen expressed by human cells.
  • This example illustrates one embodiment of the method according to the present invention for discriminating between primary tumor cells (including primary tumor cells residual from anticancer treatment), metastatic cells, and nonmalignant cells that may be present in a sample of body fluid.
  • primary tumor cells including primary tumor cells residual from anticancer treatment
  • metastatic cells including metastatic cells
  • nonmalignant cells that may be present in a sample of body fluid.
  • a number of commercially available flow cytometers can be used as the instrument on which is performed the method of the present invention. Desirably, the flow cytometer has a single laser source; and in a preferred
  • the single laser source is an argon laser tuned at 488 nanometers (nm) .
  • the flow cytometer is operatively connected to appropriate operating software and data management systems.
  • a sample of the body fluid to be analyzed can be processed using anyone of several methods known to those skilled in the art. If the body fluid is peripheral blood, the sample may, but it is not required to, be treated to remove erythrocytes by centrifugation, or with a lysing agent to lyse erythrocytes present in the sample.
  • a lysing agent includes 1% NH 4 C1 or other commercially available lysing solutions (for a review, see Tiirikainen, 1995, Cytometry, 20:341-8).
  • erythrocytes may be removed prior to loading the sample to be analyzed onto the flow cytometer.
  • a large range of light scatter signals is obtained using unlysed samples. Since platelets, erythrocytes and cell debris are typically less than 8 microns (m) , and primary tumor cells and metastatic cells are typically 8-20m or larger, forward and side light scatter signals can be gated for size and/or to nucleated events (e.g. to exclude platelets, erythrocytes and cell debris from subsequent analysis) . In that regard, nucleated events were gated by setting the instrumental threshold to exclude smaller cells and debris.
  • the sample may be prepared for analysis using any one of a number of methods for sample preparation for flow cytometric analysis known to those skilled in the art.
  • the sample may be adjusted for cell density by diluting the sample in a physiological buffer (e.g., phosphate buffered saline (PBS) ; PBS with 1% bovine serum albumin).
  • PBS phosphate buffered saline
  • the sample preferably containing 100,000 to 200,000 cells in a small volume (e.g., ranging from 20ml to 100ml) , was incubated with at least two different pretitered detector molecules for 20-30 minutes at 4°C. After this
  • cells in the sample do not need to be fixed. However, if desired, cells in the sample may be fixed prior to staining with detector molecules and may be permeabilized by adding a non- ionic detergent (for detecting cell molecules in the cytoplasm and on the cell surface) , or after staining with detector molecules (for detecting cell surface molecules).
  • Cells can be fixed by incubating them with any one of a number of solutions known in the art to include, but are not limited to, 1% paraformaldehyde, methanol, methanol/acetone, acetone, 2% (v/v) paraformaldehyde and acetone, and 70% ethanol .
  • a first detector molecule comprised PE-conjugated mAb against IL-2Ra for detecting cell molecule IL-2Ra
  • a second detector molecule comprised FITC-conjugated mAb against Ber-EP4 for detecting cell molecule epithelial cell antigen.
  • an aliquot of the sample can be incubated with the appropriate detectable moiety labelled IgG isotype controls.
  • the sample is then analyzed on a flow cytometer by exciting the fluorescence using an argon laser at 488 nm.
  • the fluorescent signals were collected through a 530 nm band pass filter for the FITC emissions, and a 585 nm band pass filter for the PE emissions.
  • forward scattering and side scattering are measured.
  • forward scatter (FS) and side scatter (90°LS) may be used to gate nucleated events (i.e., select for the size and granularity representative of nucleated cells).
  • FS forward scatter
  • side scatter 90°LS
  • BER-EP4 fluorescence BER-EP4 fluorescence
  • FIG. 4 illustrates that the level of fluorescence background events ("Events") detected, in analysis of a negative control comprising a PE-labelled IgG isotype matched antibody, is 10 1 (note width of marker bar in FIG. 4)
  • 10 1 a threshold
  • a quadrant was applied to the histogram depicted in FIG. 3 to provide for accurate enumeration of Ber-EP4+/IL-2Ra+ cells.
  • the events present in the upper right quadrant of FIG. 3 represent the Ber-EP4+/IL-2Ra+ cells.
  • gatings, and thresholds allows for a small margin of error. In this illustration (FIGs. 1-4), the margin of error was 0.03%.
  • Ber-EP4 16 lyzed, no significant (99% confidence level) staining with Ber-EP4 was detected. This supports the reported selective expression of Ber-EP4 as being cells of epithelial origin, while excluding all lymphatic cells, endothelial cells, and hematopoietic cells.
  • tissue biopsy aspirates were obtained from thirteen women ranging in ages from 39 years old to 63 years old with a clinical history of breast cancer.
  • Neoplastic disease included infiltrating ductal carcinoma, extensive ductal carcinoma, and intraductal and infiltrating carcinoma ("CA", Table 2).
  • tissue biopsy aspirates were obtained from lymph nodes regional to a carcinoma ("LN", Table 2) .
  • the preparation, staining, and flow cytometry set-up procedures were performed as described in Example 1. Some of the samples had also been analyzed by histochemical staining using hematoxylin and eosin, and visualization by light microscopy.
  • tissue biopsy aspirates were analyzed for differentially detecting and enumerating metastasizing cells.
  • the preparation, staining, and flow cytometry set-up procedures were performed as described in Example 1.
  • Case #1 Previous cancer was a grade I melanoma, with no recurrence reported. Six years later, an adenopathy raised concern for recurrence. The pathology report indicated that it was a benign process with no tumor involvement. A sample of a lymph node aspirate which was analyzed according to the present invention showed 0.00% Ber-EP4+/IL-2Ra + cells. This is consistent with selective expression of Ber-EP4 as being cells of epithelial origin, and not melanoma cells.
  • Case #3 Reported was a poorly differentiated breast carcinoma. Twenty axillary lymph nodes were obtained in surgery; and the pathology report indicated metastasis. A sample of a tumor aspirate which was analyzed according to the present invention showed 18.0% Ber-EP4+/IL-2Ra + cells. Samples of two different lymph node aspirates which were analyzed according to the present invention showed 3.1% Ber- EP4+/IL-2Ra + cells and showed 10.2% Ber-EP4+/IL-2Ra + cells, respectively.
  • Case #4 Previously reported was a intraductal breast carcinoma, with recurrence reported. Tumor biopsy was obtained in surgery; and the pathology report indicated metastasis. A sample of an biopsy aspirate which was analyzed according to the present invention showed 48.6%
  • a sample of a tumor aspirate which was analyzed according to the present invention showed 19.0% Ber-EP4+/IL-2Ra + cells.
  • a sample of a lymph node aspirate which was analyzed according to the present invention showed 2.9% Ber-EP4+/IL-2Ra + cells .
  • a sample of a single lymph node aspirate which was analyzed according to the present invention showed 12.0% Ber-EP4+/IL-2Ra + cells.
  • Case #7 Reported was intraductal breast carcinoma. Nineteen axillary lymph nodes were obtained in surgery; and the pathology report indicated metastasis in four of the nodes.
  • a sample of a the biopsy aspirate which was analyzed according to the present invention showed 2.9% Ber-EP4+/IL- 2Ra + cells.
  • Case #8 Reported was intraductal breast carcinoma. Forty one axillary lymph nodes were obtained in surgery; and the pathology report indicated no metastasis. A sample of the biopsy aspirate which was analyzed according to the present invention showed 2.9% Ber-EP4+/IL-2Ra + cells.
  • Concomitant use of the epithelial cell antigen and IL-2Ra as markers, may be useful in establishing a sensitive and statistically appropriate alternative standard for evaluating progression or recur- rence of tumor in patients. For example, because of the problems that metastases present, in terms of diagnosis and treatment of non-lymphoid tumors, a method for differentially detecting metastatic cells is desirable.
  • detection of both epithelial cell antigen and IL-2Ra markers correlates with the percentage or number of metastatic cells present in a sample, and thus may be used for determining the staging of tumor progression, and the patient outcome (i.e., a large number of cells in a sample which stain positive for both epithelial cell antigen and IL2Ra is associated with a poorer prognosis) and/or may be an indicator that metastatic recurrent has occurred.
  • the number of cells detected in each measurement are then compared to determine if there is an increase (an indicator of advancing stages, and a poorer prognosis), a decrease (an indicator of a decrease in malignant disease) , or a constant level (maintaining the same stage of malignant disease) .
  • a method for determining the staging of tumor progression e.g., by detecting and enumerating metastatic cells in a sample)
  • a clinical diagnostic kit for determining epithelial cell antigen and IL-2Ra expression.
  • a clinical diagnostic kit may comprise, for example a detector molecule having binding specificity for human IL-2Ra, a detector molecule having binding specificity for an epithelial cell antigen expressed by human cells, wherein the detectable moiety on the detector molecule having binding specificity for human IL- 2Ra has a peak emission spectrum which is distinguishable from the peak emission spectrum of the detectable moiety on the detector molecule having binding specificity for an epithelial cell antigen expressed by human cells.

Abstract

L'invention concerne une nouvelle méthode de détection et de dénombrement différentiels de cellules métastatiques présentes dans un échantillon de fluide biologique complexe, la méthode consistant à obtenir un échantillon de liquide organique provenant d'un individu; à ajouter à l'échantillon au moins deux molécules de détection, une première molécule de détection présentant une spécificité de liaison à une molécule cellulaire comprenant IL-2Ra, et une deuxième molécule de détection présentant une spécificité de liaison à une molécule cellulaire comprenant un antigène de cellule épithéliale et la fraction détectable de la première molécule de détection possédant un spectre d'émission maximal pouvant être distingué du spectre d'émission maximal de la fraction détectable de la deuxième molécule de détection; puis à analyser les cellules présentes dans l'échantillon dans un cytomètre de flux capable de détecter et de distinguer les spectres d'émission des première et deuxième molécules de détection et la diffusion de lumière de chaque cellule présente dans l'échantillon. L'invention concerne également une trousse de diagnostic clinique comprenant une molécule de détection présentant une spécificité de liaison avec IL-2Ra humain, et une molécule de détection présentant une spécificité de liaison avec un antigène de cellule épithéliale exprimé par des cellules humaines, la fraction détectable de la molécule de détection qui présente une spécificité de liaison avec IL-2Ra humain, possédant un spectre d'émission maximal pouvant être distingué du spectre d'émission maximal de la fraction détectable de la molécule de détection possédant une spécificité de liaison avec un antigène de cellule épithéliale exprimé par des cellules humaines. Le dessin constitue un histogramme représentant un antigène de cellule épithéliale (Ber-EP4 + cellules à fonctionnement commandé (FS)) et IL-2Ra + cellules (CD25).
PCT/US1999/004005 1998-02-27 1999-02-24 Methode et compositions de detection differentielle de cellules tumorales et de cellules metastatiques WO1999044064A1 (fr)

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003023057A2 (fr) * 2001-09-06 2003-03-20 Adnagen Ag Procede et kit de diagnostic destines a la selection et/ou detection qualitative et/ou quantitative de cellules
WO2003023060A2 (fr) * 2001-09-06 2003-03-20 Adnagen Ag Methode et trousse de diagnostic ou de surveillance du traitement du cancer du sein
WO2003023059A2 (fr) * 2001-09-06 2003-03-20 Adnagen Ag Procede et necessaire destines au diagnostic ou au controle du traitement du cancer de l'intestin
US7276170B2 (en) 2002-02-04 2007-10-02 Colorado School Of Mines Laminar flow-based separations of colloidal and cellular particles
USRE41762E1 (en) 2001-02-14 2010-09-28 Stc.Unm Nanostructured separation and analysis devices for biological membranes
US8137912B2 (en) 2006-06-14 2012-03-20 The General Hospital Corporation Methods for the diagnosis of fetal abnormalities
US8168389B2 (en) 2006-06-14 2012-05-01 The General Hospital Corporation Fetal cell analysis using sample splitting
US8195415B2 (en) 2008-09-20 2012-06-05 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnosis of fetal aneuploidy by sequencing
US8585971B2 (en) 2005-04-05 2013-11-19 The General Hospital Corporation Devices and method for enrichment and alteration of cells and other particles
US8895298B2 (en) 2002-09-27 2014-11-25 The General Hospital Corporation Microfluidic device for cell separation and uses thereof
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US9487812B2 (en) 2012-02-17 2016-11-08 Colorado School Of Mines Optical alignment deformation spectroscopy
US9878326B2 (en) 2007-09-26 2018-01-30 Colorado School Of Mines Fiber-focused diode-bar optical trapping for microfluidic manipulation
US9885644B2 (en) 2006-01-10 2018-02-06 Colorado School Of Mines Dynamic viscoelasticity as a rapid single-cell biomarker
US10591391B2 (en) 2006-06-14 2020-03-17 Verinata Health, Inc. Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US10704090B2 (en) 2006-06-14 2020-07-07 Verinata Health, Inc. Fetal aneuploidy detection by sequencing
US10722250B2 (en) 2007-09-04 2020-07-28 Colorado School Of Mines Magnetic-field driven colloidal microbots, methods for forming and using the same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4284412A (en) * 1979-07-13 1981-08-18 Ortho Diagnostics, Inc. Method and apparatus for automated identification and enumeration of specified blood cell subclasses
US4727020A (en) * 1985-02-25 1988-02-23 Becton, Dickinson And Company Method for analysis of subpopulations of blood cells
US5536642A (en) * 1993-09-09 1996-07-16 Barbera-Guillem; Emilio Diagnostic and prognostic methods for solid non-lymphoid tumors and their metastases
US5569585A (en) * 1993-03-12 1996-10-29 Cellcor, Inc. In vitro assay measuring degree of activation of immune cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4284412A (en) * 1979-07-13 1981-08-18 Ortho Diagnostics, Inc. Method and apparatus for automated identification and enumeration of specified blood cell subclasses
US4727020A (en) * 1985-02-25 1988-02-23 Becton, Dickinson And Company Method for analysis of subpopulations of blood cells
US5569585A (en) * 1993-03-12 1996-10-29 Cellcor, Inc. In vitro assay measuring degree of activation of immune cells
US5536642A (en) * 1993-09-09 1996-07-16 Barbera-Guillem; Emilio Diagnostic and prognostic methods for solid non-lymphoid tumors and their metastases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BARTONI D P J, ET AL.: "EXPRESSION OF INTERLEUKIN-2 RECEPTOR ALPHA (IL-2RALPHA) MRNA AND PROTEIN IN ADVANCED EPITHEHAL OVARIAN CANCER", ANTICANCER RESEARCH - INTERNATIONAL JOURNAL OF CANCER RESEARCH AND TREATMENT, INTERNATIONAL INSTITUTE OF ANTICANCER RESEARCH, GR, vol. 14, 1 January 1994 (1994-01-01), GR, pages 761 - 772, XP002918828, ISSN: 0250-7005 *

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