WO2003019141A2 - Analysis of circulating tumor cells, fragments, and debris - Google Patents
Analysis of circulating tumor cells, fragments, and debris Download PDFInfo
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- WO2003019141A2 WO2003019141A2 PCT/US2002/026861 US0226861W WO03019141A2 WO 2003019141 A2 WO2003019141 A2 WO 2003019141A2 US 0226861 W US0226861 W US 0226861W WO 03019141 A2 WO03019141 A2 WO 03019141A2
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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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
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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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
- G01N33/543—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
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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
Definitions
- CTC circulating tumor cells
- 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 for breast cancer, or measurements of Prostate Specific Antigen (PSA) for prostate cancer.
- PSA Prostate Specific Antigen
- the organ origin of such cells may readily be determined, e.g., breast, prostate, colon, lung, ovarian or other non- hematopoietic cancers.
- Malignant tumors are characterized by their ability to invade adjacent tissue.
- tumors with a diameter of 1mm 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.
- 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).
- Increase in tumor mass might be expected to be proportional to an increase in the frequency of CTC, as single cells, or clusters of cells with increased adhesiveness (and possibly greater invasive potential).
- 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.
- whole blood is a complex body fluid containing diverse populations of cellular and soluble components capable of undergoing numerous biochemical and enzymatic reactions in vivo and in vitro, particularly on prolonged storage for more than 24hrs. Some of these reactions are related to immunological destruction of CTC that are recognized as foreign species.
- the patient's immune response weakens or destroys tumor cells by the normal defense mechanisms including phagocytosis and neutrophil activation.
- Chemotherapy similarly is intended to reduce both cell function and proliferation by inducing cell death by necrosis. Besides these external destructive factors, tumor cells damaged in a hostile environment may undergo programmed death or apoptosis.
- Normal and abnormal cells including CTC undergoing apoptosis or necrosis, have altered membrane permeabilities that allow escape of DNA, RNA, and other intracellular components leading to formation of damaged cells, fragmented cells, cellular debris, and eventual complete disintegration.
- tumor cell debris may still bear epitopes or determinants characteristic of intact cells and can lead to spurious increases in the number of detected circulating cancer cells.
- Whole blood specimens from healthy individuals also have been observed to undergo destruction of labile blood cell components, herein categorized as decreased blood quality, on prolonged storage for periods of greater than 24 hours. For example, erythrocytes may rupture and release hemoglobin and produce cell ghosts.
- Leukocytes particularly granulocytes, are known to be labile and diminish on storage.
- Such changes increase the amount of cellular debris that can interfere with the isolation and detection of rare target cells such as CTC.
- the combined effects of these destructive processes can substantially increase cellular debris, which is readily detectable, for instance, in flow cytometric and microscopic analyses, such as CellSpotter® or CellTracksTM.
- Detection of circulating tumor cells by microscopic imaging is similarly adversely affected by spurious decreases in classifiable tumor cells and a corresponding increase in interfering stainable debris.
- maintaining the integrity or the quality of the blood specimen is of utmost importance, since there may be a delay of as much as 24 hours between blood draw and specimen processing.
- Such delays are to be expected, since the techniques and equipment used in processing blood for this assay may not be readily available in every laboratory.
- the time necessary for a sample to arrive at a laboratory for sample processing may vary considerably. It is therefore important to establish the time window within which a sample can be processed. In routine hematology analyses, blood samples can be analyzed within 24 hours.
- the time window in which a blood sample can be analyzed is shorter.
- An example is immunophenotyping of blood cells, which, in general, must be performed within 24 hours.
- larger volumes of blood have to be processed, and in vitro degradation of the blood sample can become more problematic as materials released by disintegrating cells, both from CTC and from hematopoietic cells, can increase the background and therefore decrease the ability to detect tumor cells.
- CTC can be considered immunologically foreign species, normal cellular immune responses of the host will occur in vivo even before blood draw. Also large numbers of CTC can be continuously shed from a tumor site, and a steady-state level is maintained in which destruction of CTC equals the shedding rate which in turn depends on the size of the tumor burden (see JG
- Chen describes the use of an adhesion matrix for enumerating tumor cells. Briefly, the matrix is coated with specific adhesion molecules that will bind viable cancer cells with presumed metastatic potential. The matrix can then be analyzed for the presence and type of captured cells. Also described are methods for using the matrix in screening treatments. While steps are taken to discriminate between intact cells and apoptotic or necrotic cells, the apoptotic or necrotic cells are specifically excluded from analysis.
- WO00/47998 from Cell Works, two pathways are described for CTC, terminal and proliferative. Both pathways begin with an "indeterminate" cell that progresses, as determined by morphological differences, down either the terminal or proliferative pathway. A cell in the terminal pathway eventually is destroyed, and a cell in the proliferative pathway will form a new metastatic colony as a metastatic tumor. This proliferative pathway (cells and clusters) is the focus as a potential diagnostic, but minimizes the significance of cell fragments or debris. These two pathways were designed to explain morphological differences seen in patient samples.
- Apoptosis is characterized by a series of stepwise slow intracellular events, which differs from necrosis or rapid cell death triggered or mediated by an extracellular species, e.g. a cytotoxic anti-tumor drug.
- CTC can circulate as both live and dead cells, wherein "dead” comprises the full range of damaged and fragmented cells as well as CTC-derived debris.
- the tumor burden is probably best represented by the total of both intact CTC, including clusters, and damaged CTC, which bear morphological characteristics of cells, but are distinct from clumps and/or aggregates.
- CTC debris that is positively stained for cytokeratin may also have densities falling in the RBC or higher ranges, since most intracellular components (with the possible exception of lipophilic membrane fragments that may be located near the plasma-buffy coat interface) have densities in the range of 1.15 to 1.3. Hence, a substantial portion of damaged CTC and CTC debris may be located outside the buffy coat layer, and would not be seen by the density gradient methods, such as those in WO00/47998. Some images of damaged or fragmented CTC are shown, but it is quite possible the damage occurred during cytospin or subsequent processing, and is thus artifactual. While the densities of most intact tumor cells may fall in the WBC region, it is quite likely that damaged CTC in patient samples have higher densities that may place them in the RBC layer; outside the reach of gradient techniques.
- Epithelial cells in their tissue of origin obey established growth and development "rules". Those rules include population control. This means that under normal circumstances the number and size of the cells remains constant and changes only when necessary for normal growth and development of the organism. Only the basal cells of the epithelium or immortal cells will divide and they will do so when it is necessary for the epithelium to perform its function, whatever it is depending in the nature and location of the epithelium. Under some abnormal but benign circumstances, cells will proliferate and the basal layer will divide more than usual, causing hyperplasia. Under some other abnormal but benign circumstances, cells may increase in size beyond what is normal for the particular tissue, causing cell gigantism, as in folic acid deficiency.
- Epithelial tissue may increase in size or number of cells also due to pre-malignant or malignant lesions. In these cases, changes similar to those described above are accompanied by nuclear abnormalities ranging from mild in low-grade intraepithelial lesions to severe in malignancies. It is believed that changes in these cells may affect portions of the thickness of the epithelium and as they increase in severity will comprise a thicker portion of such epithelium. These cells do not obey restrictions of contact inhibition and continue growing without tissue controls. When the entire thickness of the epithelium is affected by malignant changes, the condition is recognized as a carcinoma in situ (CIS).
- CIS carcinoma in situ
- the malignant cells eventually are able to pass through the basement membrane and invade the stroma of the organ as their malignant potential increases. After invading the stroma, these cells are believed to have the potential for reaching the blood vessels. Once they infiltrate the blood vessels, cells find themselves in a completely different environment from the one they originated from.
- the cells may infiltrate the blood vessels as single cells or as clusters of two or more cells.
- a single cell of epithelial origin circulating through the circulatory system is destined to have one of two outcomes. It may die or it may survive.
- Single Cells :
- the cell may die either through apoptosis due to internal changes or messages in the cell itself. These messages may have been in the cell before intravasation or they may be received while in the blood, or it may die due to the influence of the immune system of the host, which may recognize these cells as "alien" to this environment.
- the results of cellular death are identifiable in CellSpotter® as enucleated cells, speckled cells or amorphous cells. These cells do not have the potential for cell division or for establishing colonies or metastases.
- Enucleated cells are the result of nuclear disintegration and elimination (karyorrhexis and karyolysis). They are positive for cytokeratin, and negative for nucleic acid.
- speckled cells are positive for cytokeratin and DAP! and show evidence of cellular degeneration and cytoplasmic disintegration. These cells may represent response to therapy or to the host's immune system as the cytoskeletal proteins retract.
- CellSpotter® Another dying tumor cell identifiable using CellSpotter® is the amorphous cell. These cells are probably damaged during the preparation process, a sign that these may be weaker, more delicate cells but may also be the result of apoptosis or immune attack. 2. A viable malignant epithelial cell may have the potential to survive the circulation and form colonies in distant organs. These "survivor cells" appear in CellSpotter® as intact cells with high nuclear material/cytoplasmic material ratio.
- Clusters The primary tumor may shed clusters that enter the circulation as described by B Brandt et al. ("Isolation of prostate-derived single cells and cell clusters from human peripheral blood.” Cancer Research 56 p4556-4561. 1996). These clusters may remain as clusters and invade a distant tissue or they may become dissociated in the circulation, probably due to differences in pressure in blood or to the immune system's intervention. If these cells are dissociated into single cells, they may follow one of the two paths described for single cells above (see 1 and 2). Cluster formations may have an effect in survival by using the outside cells as a shield that protects the inner cells from the immune system.
- a decrease in the number of tumor cells and/ or increase in the response index may represent a response to patient therapy.
- CellSpotter® can be used to help in the decision tree of these patients. All patients with an abnormal pap (5-10% of the pap smears in the USA) can immediately be tested for circulating epithelial cells. Patients with positive tests should be followed-up immediately and aggressively. Patients with negative results may wait the three months for the repeat pap. This would simplify the decision making process for the physician and health professionals and help the patient trust her follow-up procedure. Screening: CellSpotter® image analysis may be used for screening of the general population with the condition that special, tissue specific antibodies would be used on a second test on all abnormal samples.
- Identification of CTC in a patient may indicate that there is a primary malignancy that has started or is starting the process of metastasis. If these cells are identified as of the tissue of origin with new markers, then organ specific tests, like CT guided fine needle aspirations (FNA) can be used to verify the presence or absence of such malignancies. Patients where a primary cannot be identified may be followed-up with repeat tests after establishing an individual base line.
- FNA CT guided fine needle aspirations
- the number of intact CTC, damaged or Suspect CTC as well as the degree of damage to the CTC, may further serve as diagnostically important indicators of the tumor burden, the proliferative potential of the tumor cells and/or the effectiveness of therapy.
- the methods and protocols of the prior art combine unavoidable in vivo damage to CTC with avoidable in vitro storage and processing damage, thus yielding erroneous information on CTC and tumor burdens in cancer patients.
- the relatively simple blood test of the present invention described herein, which functions with a high degree of sensitivity and specificity, can be thought of as a "whole body biopsy.”
- the methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and the CellSpotter fluorescent microscopy imaging system.
- the Examples show the importance of not only analyzing Obvious or Intact CTC, but Suspect CTC or damaged fragments, clusters of CTC, and debris. It is possible to mimic the damage that forms fragments and debris. It is also possible to inhibit further damage of CTC between the blood draw and sample processing through the use of stabilizing agents.
- kits for assaying biological specimens using these methods are also provided.
- Figure 1 Models of tumor shedding and metastasis, la. shows possible stages of cells, clusters, and fragments, lb. shows the same model with actual images from samples.
- Figure 2 Flow cytometric analysis of immunomagnetically enriched tumor cells from 7.5ml blood.
- Figure 3 CellSpotter analysis of a 7.5ml blood sample from a metastatic prostate cancer patient that was immunomagnetically enriched for tumor cells. The lines of thumbnails correspond to the different dyes used in the staining process showing tumor candidates stained with cytokeratin PE (green) and DAPI (magenta).
- Figure 4 - CellSpotter ® classifications of tumor cells isolated from a single whole blood sample of a patient with metastatic prostate cancer stained with cytokeratin PE (green) and DAPI (magenta).
- Figure 5 A comparison of the number of Obvious CTC to Suspect CTC in 20 clinical samples.
- Figure 6 CellSpotter ® classifications of paclitaxel treated LnCaP cells spiked into whole blood and isolated then stained with cytokeratin PE (green) and DAPI (magenta). A - intact cells
- Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, by centrifugation of finely divided particles or beads coupled to the target substance.
- Such particles or beads may be made magnetic to facilitate the bound/free separation step.
- Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions.
- any material that facilitates magnetic or gravitational separation may be employed for this purpose.
- magnetic separation means are the method of choice.
- 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.
- Nanoparticles also known as ferrofluids or ferrofluid-like materials, have many of the properties of classical ferrofluids, and are sometimes referred to herein as colloidal, superparamagnetic particles.
- 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. 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;
- NBS non-specific binding
- the level of non-specific binding of a system is substantially constant, as is usually the case, then as the target population decreases so will the purity. Less obvious is the fact that the smaller the population of a targeted cell, the more difficult it will be to magnetically label and to recover. Furthermore, labeling and recovery will markedly depend on the nature of magnetic particle employed. For example, when cells are incubated with large magnetic particles, such as Dynal beads, cells are labeled through collisions created by mixing of the system, as the beads are too large to diffuse effectively.
- the probability of labeling target cells will be related to the number of magnetic particles added to the system and the length of time of mixing. Since mixing of cells with such particles for substantial periods of time would be deleterious, it becomes necessary to increase particle concentration as much as possible. There is, however, a limit to the quantity of magnetic particles that can be added, as one can substitute a rare cell mixed in with other blood cells for a rare cell mixed in with large quantities of magnetic particles upon separation. The latter condition does not markedly improve the ability to enumerate the cells of interest or to examine them.
- 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, and their stability to gravitational separation from solution for extended periods of time. In addition to the many other advantages, this size range makes individual particles essentially invisible to analytical techniques commonly applied to cell analysis. Particles within the range of 90-150nm 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, glycproteins, 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 #4,795,698, #5,597,531, and #5,698,27, each incorporated by reference herein.
- the nonspecific binding of cells from whole blood with these particles was 0.3%, which is significantly better than those, produced from '531.
- from 10ml of whole blood there would be about 200,000 non-target cells that would also be isolated with the cells targeted for enrichment.
- base coated particles of mean diameter lOOnm can be produced which contain at best trace amounts of material smaller than 80nm or over 130nm.
- material of about 120nm can be made with no appreciable material smaller than 90-95nm and over 160nm.
- Such materials performed optimally with regard to recovery and could be made sub-optimal by the inclusion of 60-70nm nanoparticles.
- the preferred particle size range for use in practicing this invention is 90-150nm for base coated magnetic particles, e.g., BSA-coated magnetite.
- 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 were identified by the expression of the cytoskeletal proteins cytokeratin (CK+), the absence of the common leukocyte antigen CD45 (CD45-) and the presence of nucleic acids (NA+) by multicolor fluorescence analysis. Rare events or rare cells can be immunophenotyped by both flowcytometry and fluorescence microscopy.
- CK+ cytoskeletal proteins cytokeratin
- CD45- common leukocyte antigen CD45
- NA+ nucleic acids
- Flowcytometric analysis excels in its ability to reproducibly quantify even low levels of fluorescence whereas microscopy has the better specificity as morphological features can aid in the classification of the immunophenotypically identified objects.
- biological specimen or “biological sample” may be used interchangeably, and refer to a small potion of fluid or tissue taken from a human test subject that is suspected to contain cells of interest, and is to be analyzed.
- a biological specimen refers to the fluidic portion, the cellular portion, and the portion containing soluble material.
- Biological specimens or biological samples include, without limit bodily fluids, such as peripheral blood, tissue homogenates, nipple aspirates, colonic lavage, sputum, bronchial (alveolar) lavage, pleural fluids, peritoneal fluids, pericardial fluids, urine, and any other source of cells that is obtainable from a human test subject.
- An exemplary tissue homogenate may be obtained from the sentinel node in a breast cancer patient.
- the term "rare cells” is defined herein as cells that are not normally present in biological specimens, but may be present as an indicator of an abnormal condition, such as infectious disease, chronic disease, injury, or pregnancy. Rare cells also refer to cells that may be normally present in biological specimens, but are present with a frequency several orders of magnitude less than cells typically present in a normal biological specimen.
- a “biospecific ligand” or a “biospecific reagent,” used interchangeably herein, may specifically bind determinants.
- a determinant 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.
- 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.
- 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).
- Analysis can be performed using any of a number of commonly used platforms, including multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, CellSpotter® analysis, CellTracksTM analysis, and automated cell analysis.
- 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 non-specific binding to other sample components.
- 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. In cases where peripheral blood is used as the starting materials, 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.
- anti-coagulant or “anti-coagulating agent” may be used interchangeably, and refer to compositions that are added to biological specimens for the purpose of inhibiting any undesired natural or artificial coagulation.
- coagulation is blood clotting and common anti-coagulants are chelating agents, exemplified by ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,2-diaminocyclohexane tetraacetic acid (DCTA), ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA), or by complexing agents, such as heparin, and heparin species, such as heparin sulfate and low- molecular weight heparins. This may be further collectively defined as "clumping' or "clump formation". However, such clumps must be differentiated from "clusters" or aggregates of CTC that are counted as a single Intact CTC if they meet the classification criteria for Intact CTC.
- EDTA ethylenediamine tetraacetic acid
- DTPA diethylenetriamine pentaacetic acid
- DCTA 1,
- Clusters of CTC are believed to have greater proliferative potential than single CTC and their presence is thus diagnostically highly significant.
- One possible cause for an increased propensity to establish secondary metastatic tumor sites may be the virtue of their adhesiveness.
- An even more likely cause is the actual size of a CTC cluster; larger clusters will become lodged in small diameter capillaries or pores in bone. Once there, the viability of the cells in the cluster would determine the chance of survivability at the new metastatic site.
- the ideal "stabilizer” or “preservative” is defined as a composition capable of preserving target cells of interest present in a biological specimen, while minimizing the formation of interfering aggregates and cellular debris in the biological specimen, which in any way can impede the isolation, detection, and enumeration of targets cells, and their differentiation from non-target cells.
- a stabilizing agent when combined with an anti-coagulating agent, a stabilizing agent should not counteract the anti-coagulating agent's performance. Conversely, the anti-coagulating agent should not interfere with the performance of the stabilizing agent.
- the disclosed stabilizers also serve a third function of fixing, and thereby stabilizing, permeabilized cells, wherein the expressions "permeabilized” or “permeabilization” and “fixing”, “fixed” or “fixation” are used as conventionally defined in cell biology.
- stabilizing agents herein implies using these agents at appropriate concentrations or amounts, which would be readily apparent to one skilled in cell biology, where the concentration or amount is effective to stabilize the target cells without causing damage.
- concentration or amount is effective to stabilize the target cells without causing damage.
- One using the compositions, methods, and apparatus of this invention for the purpose of preserving rare cells would obviously not use them in ways to damage or destroy these same rare cells, and would therefore inherently select appropriate concentrations or amounts.
- the formaldehyde donor imidazolidinyl urea has been found to be effective at a preferred concentration of 0.1-10%, more preferably at 0.5-5% and most preferably at about 1-3% of the volume of said specimen.
- An additional agent, such as polyethylene glycol has also been found to be effective, when added at a preferred concentration of about 0.1% to about 5%, more preferably about 0.1% to about 1%, and most preferably about 0.1 % to about 0.5% of the specimen volume.
- a stabilizing agent must be capable of preserving a sample for at least a few hours. However, it has been shown that samples can be stabilized for at least up to 72 hours. Such long-term stability is important in cases where the sample is obtained in a location that is distant to the location where processing and analysis will occur. Furthermore, the sample must be stabilized against mechanical damage during transport.
- Stabilizing agents are necessary to discriminate between in vivo tumor cell disintegration and disintegration due to in vitro sample degradation. Therefore, stabilizing agent compositions, as well as methods and apparatus for their use, are described in a co- pending application entitled “Stabilization of cells and biological specimens for analysis.” That commonly owned application is incorporated by reference herein.
- Optunous cells or “intact cells” may be used interchangeably, and refer to cells found during imaging analysis that contain nucleic acid and cytokeratin. These cells are usually visually round or oval, but may sometimes be polygonal or elongated, and appear as individual cells or clusters of cells.
- the nucleic acid area i.e. labeled by nucleic acid dye
- the cytoplasmic area i.e. labeled by anti-cytokeratin
- Suspect cells may be used interchangeably, and refer to cells found during imaging analysis that resemble intact cells, but are not as visually distinct as intact cells. Based on imaging analysis, there are a number of possible types of Suspect cells, including:
- Enucleated cells which are shaped like Obvious cells, are positively stained for cytokeratin, but negative for nucleic acid; 2. Speckled or punctate cells, which are positively stained for nucleic acid, but have irregularly-stained cytokeratin; and 3. Amorphic cells, which stain positively for cytokeratin and nucleic acid, but are irregular in shape, or unusually large. These suspicious cells are of interest in this invention because they may give additional information to the nature of the CTC, as well as the patient's disease. It is possible that staining or image artifacts may be observed during analysis. For example, enucleated cells sometimes appear to have a "ghost" region where the nucleus should have stained, but the corresponding region is nucleic acid negative.
- debris refers to unclassified objects that are specifically or non-specifically labeled during processing, and are visible as images during analysis, but are distinct from intact and/or Suspect cells. For example, it has been observed that damaged cells will release nuclear material. During processing, this nuclear material may be non- specifically magnetically labeled, and subsequently labeled with the nucleic acid stain. During analysis, the magnetically labeled and stained nuclear material can be observed when it has cytokeratin still attached. There are other objects that are similarly magnetically selected and stained which appear during analysis that are classified as debris.
- morphological analysis refers to visually observable characteristics for an object, such as size, shape, or the presence/absence of certain features. In order to visualize morphological features, an object is typically non-specifically stained.
- epitopical analysis refers to observations made on objects that have been labeled for certain epitopes. In order to visualize epitopic features, an object is typically specifically stained or labeled. Morphological analysis may be combined with epitopical analysis to provide a more complete analysis of an object.
- Figure 1 is a model of various CTC stages, including shedding and metastasis.
- Figure la. shows these stages for cells, clusters, fragments, and debris.
- Figure lb. shows actual images from samples at these same stages.
- the images of cells clusters, fragments, and debris were taken from CellSpotter® analyses of patient samples.
- the images of tissue samples (Origin and Metastatic sites) were taken from elsewhere (Manual of Cytology. American Society of Clinical Pathologists Press. 1983).
- a single cell shed from a primary tumor into the blood either survives or dies in blood. If it survives, it may possibly divide in blood, or colonize at a secondary site. If the cell dies, depending on the method, the cell degrades into various types of fragments or debris.
- Another possibility is a cluster of cells is shed from a primary tumor into the blood, where it may dissociate into single cells, or remain intact, and colonize at a secondary site. If the cluster dissociates, it can behave similar to the single cell described above. If the cluster remains intact, it is more likely to for a secondary colony for the reasons described above, which includes the large diameter cluster becoming lodged in a small diameter capillary. Once lodged, if the cells are viable, the cluster would form a new tumor.
- the cytoplasmic morphology is used to determine the level of differentiation (i.e. tissue of origin). For example, cytomplasmic morphology can classify cells as squamous versus glandular.
- tissue of origin i.e. tissue of origin
- cytomplasmic morphology can classify cells as squamous versus glandular.
- specimen processing e.g. transport of the blood- tube and mixing prior to analysis.
- Such mechanical damage is additional to on-going immunological, apoptotic, and necrotic processes leading to destruction of CTC that occur in vitro in a time dependent manner.
- the immunomagnetically selected cells of the paclitaxel treated samples analyzed by CellSpotter® resembled those observed in the patient blood samples.
- Cells treated with paclitaxel displayed signs of apoptosis.
- the punctate cytokeratin staining pattern of the cells appear to correspond with a collapse of the cytoskeletal proteins ( Figure 4B vs. 6B).
- the initiating event in the sequence resulting from the microtubule stabilizing effects of paclitaxel which in turn may activate the pro-apoptotic gene Bim that senses cytoskeletal distress.
- 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.
- the immunomagnetic sample preparation is important for reducing sample volume and obtaining as much as a 10 4 fold enrichment of the target (epithelial) cells.
- the reagents used for the multi-parameter flow cytometric 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 1. 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. 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).
- Magnetic nanoparticles labeled with monoclonal antibodies identifying epithelial cell adhesion molecule were used to label and separate by magnetic means epithelial cells from hematopoietic cells, as taught in commonly-owned US Patent #6,365,362, and US Patent Application 10/079,939, filed 19 February 2002, both of which are fully incorporated by reference herein.
- EpCAM epithelial cell adhesion molecule
- a monoclonal antibody that recognizes keratins 4, 5, 6, 8, 10, 13, and 18, conjugated to Phycoerythrin (CK-PE) was used to identify epithelial cells and a monoclonal antibody that recognizes CD45 was used to identify leukocytes and identify hematopoietic cells that non- specifically bind to cytokeratin.
- CD45 For multicolor fluorescent microscopy (CellSpotter ® ) analysis CD45 was conjugated to Allophycocyanin (CD45-APC, Caltag, CA) whereas for flow cytometric analysis peridinin chlorophyll protein conjugated CD45 (CD45-PerCP, BDIS San Jose, CA) was used.
- the nucleic acid specific dye DAPI (4,6-diamidino-2-phenylindole) was used to identify and visualize the nucleus in the CellSpotter ® system and the nucleic acid dye in the Procount system (BDIS, San Jose,CA) was used to identify cells by flow cytometry.
- Samples were analyzed on a FACSCalibur flow cytometer equipped with a 488nm Argon ion laser (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 for CTC events 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. For each sample, the number of events present in the region typical for epithelial cells was multiplied by 1.25 to account for the sample volume not analyzed by flow cytometry.
- FIG. 2 Panels A, B and C shows the flow cytometric analysis of a blood sample of a patient with metastatic prostate cancer.
- Two vertical lines in Panel B illustrate the low and high boundary of nucleic acid (NAD) content of leukocytes (red dots).
- CTC candidates express Cytokeratin (CK+), lack CD45 (CD45-) and contain nucleic acids (NAD+).
- CTC candidates having NAD equal or higher than leukocytes are considered cells and are depicted black.
- CK+, CD45- events with NAD content less than leukocytes were not considered target cells and depicted blue. The blue events were clearly smaller as compared with the black colored CTC as evident by the smaller forward light scatter signals.
- the CellSpotter system consists of a microscope with a Mercury Arc Lamp, a 10X objective, a high resolution X, Y, Z stage and a four-filter cube changer. Excitation, dichroic and emission filters in each of four cubes were for DAPI 365nm/400nm/400nm, for DiOC16 480nm/ 495nm/ 510nm, for PE 546nm/ 560nm/ 580nm and for APC 620nm/ 660nm/ 700nm. Images were acquired with a digital camera connected to a digital frame grabber. The surface of the chamber is 80.2 mm 2 and 4 rows of 35 images for each of the 4 filters resulting in 560 images have to be acquired to cover the complete surface.
- 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. All the images from a sample are logged into a directory that is unique to the specific sample identification. An algorithm is applied on all of the images acquired from a sample to search for locations that stain for DAPI and CK-PE. If the staining area is consistent with that of a potential tumor cell (DAPI+, CK-PE+) the software stores the location of these areas in a database. The software displays thumbnails of each of the boxes and the user can confirm that the images represented in the row are consistent with tumor cells, or stain with the leukocyte marker CD45. The software tabulates the checked boxes for each sample and the information is stored in the database.
- Figure 3 shows examples of CellSpotter ® analysis of a blood sample from a patient with metastatic prostate cancer. Regions that potentially contain tumor cells are displayed in rows of thumbnails. The ruler in the left lower corner of the figure indicates the sizes of the thumbnails. From right to left these thumbnails represent nuclear (DAPI), cytoplasmic cytokeratin (CK-PE), control cells stained with a membrane dye (DiOC ⁇ 6 (3)) and surface CD45 (CD45-APC) staining. The composite images shown at the left show a false color overlay of the purple nuclear (DAPI) and green cytoplasmic (CK-PE) staining.
- DAPI nuclear
- CK-PE cytoplasmic cytokeratin
- CD45-APC surface CD45
- the check box beside the composite image allow the user to confirm that the images represented in the row are consistent with tumor cells and the check box beside the CD45-APC image is to confirm that a leukocyte or tumor cell stain non-specifically.
- the software detected 2761 rows of thumbnails that demonstrated staining consistent with tumor cells. Eighteen of the 2761 rows are shown in the figure labeled 1631-1640 and 1869-1876. Rows numbered 1631, 1636, 1638, 1640, and 1873-1876 are checked off and display features of CTC defined as a size greater than 4 ⁇ m, the presence of a nucleus surrounded by cytoplasmic cytokeratin staining and absence of DiOC ⁇ 6 (3) and CD45 staining.
- the cell in row 1638 is large and the one in row 1640 is significantly smaller.
- the immunophenotype of the events in rows 1634 and 1869 are consistent with tumor cells but their morphology is not consistent with intact cells.
- the thumbnails in row 1869 shows a large nucleus and speckled cytoplasmic due to retraction of cytoskeletal proteins consistent with apoptosis of the cell.
- the thumbnail in row 1634 shows a damaged cell that appears to extrude its nucleus.
- the thumbnail shown in row 1632 shows a cell that stains both with cytokeratin as well as CD45 and is either a tumor cell non- specifically binding to CD45 or a leukocyte non specifically staining with cytokeratin.
- thumbnails 1633, 1635, 1637, 1639, 1870 and 1872 shows cytokeratin staining objects that are larger that 4 ⁇ m but have no resemblance to cells.
- the cytokeratin staining objects in thumbnails 1637, 1639 and 1872 are in close proximity of a leukocyte.
- FIG. 4 displays examples of the three categories of CTC isolated from a single tube of blood of a patient with metastatic prostate cancer undergoing therapy.
- Intact tumor cells shown in Figure 3A were defined as objects larger than 4 ⁇ m with a relatively smooth cytoplasmic membrane, cytoskeletal proteins throughout the cytoplasm, and an intact nucleus encompassed within the nucleus.
- Damaged CTC shown in Figure 4B were defined as objects larger than 4 ⁇ m with speckled cytokeratin staining or ragged cytoplasmic membrane, and a nucleus associated with the cytokeratin staining.
- Tumor cell fragments shown in Figure 4C were defined as round cytokeratin staining objects larger than 4 ⁇ m with or without association of nuclear material that had no morphological resemblance to a cell.
- Table 1 were sorted by increasing number of intact CTC detected by CellSpotter . 1 ®
- the proportion of intact CTC clearly constituted the smallest fraction of CTC and ranged from 0% to 22% of all CTC (mean 4%).
- the proportion of damaged CTC ranged from 1% to 100% (mean 34%) and the CTC fragments constituted the largest portion of CTC ranging from 0% to 93% (mean 62%).
- Example 4 Mimicking cell damage by in- vitro induction of apoptosis in LnCaP cells
- CTC detected by both flow cytometry and CellSpotter are comprised of intact cells and cells of cells at various stages of disintegration.
- the apoptosis induced in vitro by paclitaxel suggests that the detected CTC in patient blood samples are undergoing apoptosis, necrosis, or in vivo damage to a varying degree caused by the treatment or therapy, mechanical damage by passage through the vascular system, or by the immune system.
- Example 5 Obvious CTC and Suspect CTC are important indicators It is important to be able to distinguish between in vivo and in vitro damage for sensitive assays, such as those described here. This is especially evident when the assay attempts to determine the effectiveness of treatments or therapies, which are known to cause in vivo cellular damage. If sample handling, processing, or analysis were to result in damaging the target cells, forming Suspect cells, fragments, or debris, the assay will not give meaningful results.
- the lOO ⁇ l assay categorizes cells based on properties such as size and staining intensity.
- Obvious CTC have bright nucleic acid staining (similar to leukocytes), positive EpCAM antigen staining and size similar to leukocytes or larger.
- Suspect CTC are any objects positive for EpCAM antibody but not characterized as Obvious CTC (i.e. dim nucleic acid, size smaller than leukocytes).
- the assay identifies objects from both categories.
- Figure 5 shows the presence of Obvious and Suspect CTC in blood as determined by the lOO ⁇ l assay.
- the Suspect CTC are not created during sample processing (in vitro damage) as the lOO ⁇ l assay is a direct assay and does not involve any separation or wash steps.
- the data above also show there is a relationship between the number of Obvious and Suspect CTC.
- the number of Suspect CTC seems to increase as the number of Obvious CTC increases.
- the slope of 2.92 indicates the proportion of Suspect CTC present in sample when compared to Obvious CTC.
- Suspect CTC are also seen in the ferrofluid-selection assay, and have properties similar to Suspect CTC detected in the blood by the direct assay. It is important to include Suspect CTC in addition to Obvious CTC in total tumor cell count.
- the Suspect CTC have lower EpCAM density compared to Obvious CTC and significance of this is not yet well understood.
- a comparison was made of Obvious and Suspect CTC by the lOO ⁇ l assay to the ferrofluid-selection assay using 7ml of blood. This data was obtained from prostate patient samples and analyzed by flow cytometry. Both Obvious and Suspect CTC increased with storage time and the trend was similar to CTC detected in the ferrofluid-selection assay, thereby validating the lOO ⁇ l assay.
- the recovery of CTC from the ferrofluid-selection assay was about 90% based on the CTC in lOO ⁇ l of blood.
- MFI Mel Fluorescence Intensity which correlates the EpCAM density
- Ratio Obvious CTC / Total CTC
- Ratios near 1.0 indicate the Total CTC are Obvious CTC
- ratios near 0.0 indicate more Suspect CTC or debris.
- Progressive indicates the lesion increasing in size
- Partial Response indicates a response to treatment where the Ratio is relatively low
- Stabilized indicates no change, or reduction in lesion size.
- a positive Change indicates an increase in the number of Intact CTC, corresponding to the progression of the disease.
- a negative Change indicates a decrease in the number of Intact CTC, or a possible increase in the number of Suspect CTC and/or debris, corresponding to a response to treatment.
- Enumeration of tumor cell debris may prove more significant in cancer diagnostics and therapeutics than detection of large proliferative cell clusters. Since debris particles in the size range, probably about l-3 ⁇ m (the size of platelets), have been observed to be present in much larger amounts than intact cells, they may constitute a separate, independent, and possibly more sensitive marker than intact tumor cells. The presence of damaged CTC may be particularly relevant in detecting early-stage cancer, when the immune system is intact and most active. Similarly, dramatic increases in debris during therapy may suggest breakdown of both circulating and tissue tumor cells (i.e. therapeutic effectiveness), paralleling the massive release of cellular components like calcium observed during tumor disintegration.
- Such debris may be detectable in blood without enrichment, or with minimal enrichment in the buffy coat layer and constitute an alternative, and potentially simpler diagnostic tool than intact cell enrichment/analysis. Since morphology is lost in CTC debris, detection could be done by flow cytometry as long as the debris is stained for the appropriate determinants, such as cytokeratin.
- damaged or fragmented CTC with or without DNA are theoretically to be expected, and therefore are not undesirable events in specimens from patients undergoing effective therapy and in untreated patients with strong immune systems.
- the ratio or % of intact CTC to total detectable events may prove to be a more useful parameter to the clinician in assessing a patient's immune system or response to therapy.
- the normal immune defenses, especially activated neutrophils also can damage or destroy CTC as foreign species by a process called "extracellular killing" even if the CTC are larger than the neutrophils. It does not seem surprising to find only a small percentage of the shed CTC as intact cells, unless the immune system is overwhelmed in the late stages of disease or therapy is ineffective.
- 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 and/or clusters, fragments, or debris.
- 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 that are recognized by available monoclonal antibodies.
- the methods and the kits of the invention may be adapted to detect such circulating endothelial cells.
- 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. It would be reasonable to expect that these rare cells will behave similarly in circulation, and that fragments and/or debris will be present in similar conditions as those described hereinabove.
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Also Published As
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EP1425294B1 (en) | 2008-07-16 |
WO2003019141A3 (en) | 2004-04-01 |
CA2457891A1 (en) | 2003-03-06 |
WO2003018757A3 (en) | 2003-09-12 |
KR20040053115A (en) | 2004-06-23 |
IL160209A0 (en) | 2004-07-25 |
DE60227678D1 (en) | 2008-08-28 |
CA2457891C (en) | 2019-10-01 |
JP2005501236A (en) | 2005-01-13 |
BR0212124A (en) | 2004-07-20 |
CA2457894C (en) | 2014-07-08 |
CN1596265A (en) | 2005-03-16 |
BR0212125A (en) | 2005-06-28 |
KR20040030984A (en) | 2004-04-09 |
EP1425294A2 (en) | 2004-06-09 |
EP1425294B2 (en) | 2012-12-12 |
AU2009200013B2 (en) | 2012-03-08 |
JP2009109514A (en) | 2009-05-21 |
ATE401574T1 (en) | 2008-08-15 |
EP1425383A4 (en) | 2005-11-09 |
WO2003018757A2 (en) | 2003-03-06 |
JP2005502863A (en) | 2005-01-27 |
CA2457894A1 (en) | 2003-03-06 |
IL160210A0 (en) | 2004-07-25 |
AU2008288601A1 (en) | 2009-04-02 |
EP1425383A2 (en) | 2004-06-09 |
CN1571634A (en) | 2005-01-26 |
AU2009200013A1 (en) | 2009-02-05 |
AU2008288601B2 (en) | 2012-05-31 |
EP1425294A4 (en) | 2005-11-16 |
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