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WO2006054991A1 - Enrichissement magnétique de cellules en circulation, fragments et débris pour protéomique et génomique par hts dans la détection de maladie - Google Patents

Enrichissement magnétique de cellules en circulation, fragments et débris pour protéomique et génomique par hts dans la détection de maladie Download PDF

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WO2006054991A1
WO2006054991A1 PCT/US2004/038608 US2004038608W WO2006054991A1 WO 2006054991 A1 WO2006054991 A1 WO 2006054991A1 US 2004038608 W US2004038608 W US 2004038608W WO 2006054991 A1 WO2006054991 A1 WO 2006054991A1
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cells
ctc
improvement
analysis
cell
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PCT/US2004/038608
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English (en)
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Gerald V. Doyle
Shawn Mark O'hara
Herman Rutner
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Immunivest Corporation
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Priority to PCT/US2004/038608 priority Critical patent/WO2006054991A1/fr
Priority to CA002587765A priority patent/CA2587765A1/fr
Publication of WO2006054991A1 publication Critical patent/WO2006054991A1/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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Definitions

  • This invention generally relates to the use of proteomic and mRNA transcript investigation as diagnostic tools as it relates to the fields of oncology and diagnostic testing. More specifically, the present invention relates to the use of proteomics and mRNA transcript profiling as a source of information in the analysis of tumor cells for early diagnosis of cancer and in predicting clinical outcomes.
  • metastases i.e. multiple widespread tumor colonies established by malignant cells that detach themselves from the site of the original tumor and travel through the body to distant sites. If a primary tumor is detected early enough, it can often be eliminated by surgery, radiation, or chemotherapy or some combination of those treatments. Because of the difficulty in detection, metastatic colonies are harder to detect and eliminate and it is often impossible to treat all of them successfully. From a clinical perspective, metastasis is considered a conclusive event in the natural progression of cancer. Moreover, the ability to metastasize is the property that uniquely characterizes a malignant tumor. Cancer metastasis comprises a complex series of sequential events.
  • the test is performed on 10 5 to 10 6 cells purified away from interfering red blood cells. This corresponds to a practical lower limit of sensitivity of one tumor cell per 0.1 ml of blood. Hence, there needs to be about 10 tumor cells in an ml of blood before a signal is detectable. As a further consideration, tumor cells are often genetically unstable. Accordingly, cancer cells having genetic rearrangements and sequence changes may be missed in a PCR assay as the requisite sequence complementation between PCR primers and target sequence can be lost.
  • a useful diagnostic test needs to be very sensitive and reliable.
  • a blood test developed to detect the presence of a single tumor cell in one ml of blood corresponds to the detection of 3000 - 4000 total cells in circulation, a number that establishes tumors in inoculated animals. Further if 3000 - 4000 circulating cells represent 0.01 % of the total cells in a tumor, then it would contain about 4 x 10 7 total cells. A tumor containing that number of cells would not be visible by any technique currently in existence. Hence, if tumor cells are shed in the early stages of cancer, a test with the sensitivity mentioned above would detect the cancer. If tumor cells are shed in some functional relationship with tumor size, then a quantitative test could assess tumor burden. The general view is that tumors are initially well confined and hence there are few if any circulating cells in early stages of disease.
  • a method for identifying those cells in circulation with metastatic potential prior to establishment of a secondary tumor is highly desirable, particularly early on in the cancer.
  • 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.
  • 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.
  • 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 1.
  • 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 imaging systems 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 DAPI 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.
  • Another dying tumor cell identifiable 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.
  • a viable malignant epithelial cell may have the potential to survive the circulation and form colonies in distant organs. These "survivor cells” appear in as intact cells with high nuclear material/cytoplasmic material ratio. These cells are probably undifferentiated and can potentially divide in blood and form small clusters (Brandt et a/. "Isolation of prostate-derived single cells and cell clusters from human peripheral blood” Cancer Research 56, 4556-4561 , 1996) that may extravasate in a distant capillary, where the cell may establish a new colony, or it may remain as a single cell until it extravasates, dividing once it establishes itself in the new tissue, starting this way a new colony.
  • a decrease in the number of tumor cells and/or some change in an appropriate index may represent a response to patient therapy.
  • the response index represents a measure of response to a patient therapy whereby
  • Image cytometry analysis is useful for screening the general population. Identification of CTC in a patient could 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 guided fine needle aspirations (FNA) can be used to verify the presence or absence of such malignancies. Patients where a primary cannot be identified can be followed-up with repeat tests after establishing an individual base line. All or some of the above-cited factors were found to contribute to debris and/or aggregate formation that have been observed to confound the detection of CTC by direct enrichment procedures from whole blood as disclosed in this invention.
  • FNA 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 present invention has a distinct advantage in that 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.
  • This relatively simple blood test described herein, which functions with a high degree of sensitivity and specificity, can be thought of as a "whole body biopsy".
  • Proteomics Incorporating a more global analysis of diagnosis, follow-up, and screening as related to protein expression is another embodiment of the present invention. Assessing global patterns of protein expression in individual cells, tissues, or body fluids, has been the basic foundation in proteomics and provide an improvement to current methods. Coupled with genetic information, protein expression in individual cells can take on several different forms based upon the nucleotide sequence, whether a splice variant occurs, or whether there is a post-translational modification. Thus, the transcription, translation, and post-translational modification of each protein define a specific biochemical function within a living cell.
  • proteomics looks at the transcripts of genomic DNA (messenger RNA) as they directly encode proteins, and that these proteins are further modified by mechanisms such as phosphorylation or glycosylation. As a consequence of this sequence of events, there are functional variations in protein expression. Thus, proteomics is a process of transcriptional profiling to determine which genes, or combination thereof, are transcribed in a particular cell type or disease state.
  • protein profiling is examined by various techniques which include two-dimensional gel electrophoresis (2D-GeI) and mass spectroscopy (MS), co-immunoprecipation, affinity chromatography, protein binding analysis, overlay analysis, using yeast in protein-protein interaction, the analysis of signal transduction and other complex cellular process, three-dimensional structure modeling and large-scale protein folding, and the incorporation of bioinformatics with proteomic data.
  • 2D-GeI two-dimensional gel electrophoresis
  • MS mass spectroscopy
  • Two-dimensional gel electrophoresis alone has several inherent problems, especially when applied in diagnosis. These include difficulties in the analysis of the gels, the insufficiency of the resolving power to separate various distinct proteins in a particular sample, and a lack of reproducibility from one gel sample to the next.
  • polypeptides are solubilized in a solution or reagent system depending upon the properties of the polypeptide (i.e. organic or inorganic solvents) and the type of MS performed (WO 93/24834 by Chait et al.).
  • Mass spectrometer analysis includes ionization (I) techniques, including but not limited to matrix assisted laser desorption (MALDI), continuous or pulsed electrospray (ESI) and related methods (IONSPRAY or THERMOSPRAY), or massive cluster impact (Cl). These ion sources are matched with detection formats including linear or non-linear reflection time- off-light (TOF), single or multiple quadropole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, LC/MS, MS/MS, and combinations thereof.
  • I ionization
  • MALDI matrix assisted laser desorption
  • ESI continuous or pulsed electrospray
  • IONSPRAY or THERMOSPRAY continuous or pulsed electrospray
  • Cl massive cluster impact
  • Matrix-assisted laser desorption/ionization time of flight mass spectrometry refers to the formation of a matrix with several small, acidic, light absorbing chemicals that is mixed in solution with the analyte in such a manner so that, upon drying on the probe element, the crystalline matrix- embedded analyte molecules are successfully desorbed (by laser irradiation) and ionized from the solid phase (crystals) into the gaseous or vapor phase and accelerated as intact molecular ions.
  • the analyte is mixed with a freshly prepared solution of the chemical matrix and placed on the inert probe element surface to air dry just before the mass spectrometric analysis (see US 5,808,300).
  • SELDI Surfaces Enhanced for Laser Desorption/lonization
  • US 6,020,208 Another general category, utilizing a sample presenting means, is Surfaces Enhanced for Laser Desorption/lonization (SELDI) and described in US 6,020,208, within which there are three (3) separate subcategories.
  • the SELDI process is directed toward a sample presenting means (i.e., probe element surface) with surface-associated (or surface-bound) molecules to promote the attachment and subsequent detachment of analyte molecules in a light-dependent manner, wherein the surface-associated molecule(s) are selected from the group consisting of photoactive (photo labile) molecules that participate in the binding (docking, tethering, or ⁇ cross linking) of the analyte molecules to the sample presenting means (by covalent attachment mechanisms or otherwise).
  • photoactive photo labile
  • the mass of the target polypeptide is then compared to the mass of a reference polypeptide of known identity.
  • MS based processes for detecting a particular nucleic acid sequence in a biological sample has been described in US 6,043,031.
  • the process is used to diagnose a genetic disease or chromosomal abnormality, a predisposition to a disease or condition, infection by a pathogenic organism, or for determining heredity.
  • Detection of the desired fragments is optimum between 7,000 to 20,000 Da obtained from tryptic digests.
  • proteomics in diagnosing the existence or predicting the development and/or progression of abnormal physiological conditions based upon the presence of proteomic materials has been previously described (US 20020260420).
  • the patient sample is prepared by isolating proteomic material with characteristics identifiable for normal and abnormal physiological conditions or associated predictive endpoints, e.g down regulation or up regulation of proteins also present in healthy individuals.
  • proteomic materials are separated to permit analysis of one or more specific proteomic materials thereby enabling the diagnostician to characterize an individual's condition as being either positively or negatively indicative of one or more abnormal physiological conditions.
  • proteomics and current methods have been applied in cancer diagnostics, such methods lack simple and efficient S/N amplification or pre- enrichment methods that would improve the sensitivity and reduce the sample processing time and cost of analysis of clinical specimens.
  • the present invention provides a tool for clinicians in the diagnosis and prognosis of disease states such as cardiovascular disorders and cancer, and provides a sensitive, simple, and efficient analysis of disease detection to complement other means of detection known in the art.
  • 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 imaging systems and mRNA transcript profiling. The examples show the importance of not only analyzing obvious or intact CTC, but suspect CTC or damaged fragments, clusters of CTC, and debris. Similar analysis is possible with endothelial cells. In this type analysis, assessing the damage that forms fragments and debris is easier.
  • 2-D Gel electrophoresis, MS, SELDI or microarray detection of cells and fragments would be used alone or in conjuction with image analysis on the enriched fraction of debris and/or cells, captured by positive selection of antibody-coupled magnetic particles.
  • the present invention also includes any specific antibody-antigen, ligand- receptor, or labeling means.
  • MS is accomplished directly on the captured ferrofluid particles or on the captured target materials after dissociation from the ferrofluid by a reversible binding reaction, such as by the dissociation of the bond between target-Mab-desthiobiotin and streptavidin on the ferrofluid with soluble biotin to liberate the Mab labeled target material.
  • the direct mode is most suited for diagnostic correlation with cell counts, clinical diagnosis and the ability to differentiate target material from ferrofluid associated proteins, as well as potential utility as a complementary or independent modality to cell imaging.
  • a second approach is to limit analysis to only MS after immunomagnetic enrichment (or non-magnetic enrichment) from separate, unprocessed specimens such as whole blood, plasma or serum.
  • This approach is without cell permeabilization, antibodies and staining reagents, incorporated with image analysis, to minimize the introduction of extraneous components that would interfere with MS analysis.
  • the one embodiment of the present invention is the enrichment of target specific cell fragments, debris, and non-particulate soluble protein. These include immune complexes which are normally present at low levels in the early stages of disease and increase as the disease progresses.
  • Incorporating proteomics in cancer detection provides additional information in the analysis of circulating rare cells if enrichment provides sufficient mass for MS detection.
  • CTC debris present during low CTC
  • capture of debris containing the same surface markers as the intact cells, followed by MS analysis provides a new platform for early cancer diagnosis.
  • monclonal antibodies as capture agents (i.e. CD 146, CD 105, CD 31 , CD 133, CD 106)
  • the present invention considers diseases associated with circulating endothelial cells and their analysis. These diseases include those relating to cardiovascular disorders.
  • proteomic and transcriptome analysis especially with the enriched cell/cell debris/cell fragment components, are utilized in methodologies for diagnosing, monitoring and screening disease.
  • Figure 1 Models of tumor shedding and metastasis.
  • 1a shows possible stages of cells, clusters, and fragments.
  • 1b. shows the same model with actual images from samples.
  • Figure 2 Flow cytometric analysis of immunomagnetically enriched tumor cells from a 7.5ml blood of a metastatic prostate patient.
  • Figure 3 Image cytometry analysis with 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 and DAPI.
  • Figure 4 Classifications of tumor cells from a whole blood sample of a patient with metastatic prostate cancer stained with cytokeratin PE and DAPI. A: intact cells B: damaged tumor cells C: tumor cell fragments.
  • Figure 5 A comparison of the number of obvious CTC and suspect CTC in 20 clinical samples.
  • Figure 6 Classification of paclitaxel treated LnCaP cells spiked into whole blood and isolated then stained with cytokeratin PE and DAPI. A: intact cells B: dying tumor cells C: tumor cell fragments Figure 7: Outline of one embodiment in a sample preparation for proteomic analysis. Detailed Description of the Invention
  • Proteomics refers to the study of proteins and their DNA messenger RNA transcripts that directly encode for them. These expressed proteins can be further modified by post-translational modification, e.g. such as phosphorylation and glycosylation that alter protein expression.
  • rare cells refers to a variety of cells, microorganisms, bacteria, and the like. Cells are characterized as rare in a sample because they are not present in normal samples of the same origin, and are several orders of magnitude lower in concentration that the typical cells in a normal sample. Embodiments of the present invention include circulating cancer cells, virally, infected cells, fetal cells in maternal circulation, or endothelial cells efficiently isolated from non-rare cells and other bioentities, using the methods and apparatus of the present invention in conjunction with previously described technology (US 6,365,362).
  • analyte refers to any atom and/or molecule; including their complexes and fragments ions.
  • biological molecules/macromolecules or “biopolymers” such analytes include but are not limited to: proteins, peptides, DNA, RNA, carbohydrates, steroids, and lipids.
  • 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.
  • biofunctional polymers e.g., proteins
  • Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, US Patent Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunological reagents. The efficiency with which magnetic separations depends on many factors.
  • the preferred magnetic particles for use in the present 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.
  • magnetic alignment and subsequent mutual attraction/repulsion of these colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, contributing to solution stability.
  • the magnetic particles are separated in high magnetic gradient external field separators, facilitating sample handling and providing 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 5,698,271 , each incorporated by reference herein.
  • 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. Additionally for magnetic separations to be successful, the magnetic particles must be specific for epitopes that are not present on hematopoetic 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.
  • microscopic examination of the CK+, CD45-, NA+ objects showed that only few of the objects appeared as intact cells. This observation agrees with other reports that showed apoptosis in a substantial portion of circulating tumor cells.
  • 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.
  • 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.
  • determinant when used in reference to any of the foregoing target bioentities, refers broadly to chemical mosaics present on macromolecular antigens that often induce an immune response. Determinants may also be used interchangeably with “epitopes”.
  • 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.
  • the term "specific binding pair” as used herein 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, image cytometry analysis, and other automated cell analysis.
  • 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.
  • target bioentities e.g., tumor cells
  • red cells are not counted when assessing the extent of enrichment.
  • circulating epithelial cells may be enriched relative to leucocytes to the extent of at least 2,500 fold, more preferably 5,000 fold and most preferably 10,000 fold.
  • 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.
  • An example of 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.
  • EDTA ethylenediamine tetraacetic acid
  • DTPA diethylenetriamine pentaacetic acid
  • DCTA 1 ,2- diaminocyclohexane t
  • clumping' This may be further collectively defined as “clumping' or “clump formation”.
  • 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.
  • 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.
  • 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.
  • inventions 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;
  • Speckled or punctate cells which are positively stained for nucleic acid, but have irregularly-stained cytokeratin;
  • Amorphic cells which stain positively for cytokeratin and nucleic acid, but are irregular in shape, or unusually large. These suspicious cells are considered in the present invention because they give additional information to the nature of the CTC, as well as the patient's disease.
  • the staining or image artifacts observed during analysis provide additional informaton. 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. This may be caused by a number of external factors, including the labeling or imaging techniques. Also, cells have been observed with "detached" nuclei.
  • 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 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 1a shows these stages for cells, clusters, fragments, and debris.
  • Figure 1b shows actual images from samples at these same stages.
  • the images of cells clusters, fragments, and debris were taken from patient samples after immunomagnetic enrichment and image cytometry.
  • 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.
  • Nuclear morphology is used to determine the activity status and abnormality of a cell. Chromatin clumping, the presence or absence of nucleoli, and hyperchromasia, are criteria used to determine whether a cell is benign or malignant, reacting to an immune response, or reacting to treatment. 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.
  • the surviving battered tumor cells present in the peripheral circulation may be further stressed and damaged by turbulence during blood draw into an evacuated tube and by specimen processing, e.g. transport of the blood tube and mixing prior to analysis.
  • 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 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.
  • Further evidence of caspase-cleaved cytokeratin resulting from apoptosis was obtained with the M30 Cytodeath antibody (Roche Applied Science, Mannheim, Germany) that recognizes an epitope of cytokeratin 18 that is only exposed following caspase cleavage in early apoptosis. Only the paclitaxel treated LnCaP cells stained with M30 and most of the dimmer cytokeratin cells stained with M30, which is consistent with cells undergoing apoptosis.
  • the present invention utilizes this approach to provide clues in the early diagnosis of cancer and in prediction of clinical outcomes.
  • One of the biggest problems in the clinical use of this approach is the selective extraction or enrichment of the desired global target entities, which typically number fewer than 100, from highly complex samples containing millions of irrelevant entities.
  • MS mass spectrometry
  • SELDI-TOF Laser Desorption/lonization Time-Of-Fight
  • non-magnetic affinity-based solid phase separation can also be used to selectively enrich specific targets or target populations (e.g. antibody coated particles or solid phases for capturing the target materials, followed by analysis of the enriched fraction without or with prior dissociation from the support).
  • targets or target populations e.g. antibody coated particles or solid phases for capturing the target materials, followed by analysis of the enriched fraction without or with prior dissociation from the support.
  • Magnetic separation with ferrofluid particles described in US 6,365,362 provides a means of enrichment that is inexpensive and simple. Further, these ferrofluid particles provide higher binding capacities than other larger particles or non-magnetic solid phase particles (e.g. gel particles).
  • the one embodiment of the present invention uses the procedure described in US 6, 365,362 to incorporate multiparametric image cytometry and morphological characterization of selectively stained tumor cells together with proteomic analysis in cancer diagnosis.
  • magnetic enrichment of rare target cells, along with associated cell fragments and debris are coupled with proteomics as an alternative means of cancer cell detection.
  • Magnetic enrichment of rare target cells can occur after pretreatment with or without preservative (U.S. application 10/780,399).
  • immunomagnetic (or alternatively non-magnetic) enrichment pathological cells, cell fragments, debris, and soluble cell fractions from patient specimens are assessed by MS, SELDI, microchips, biochips, or multiplexed micro array analysis. The detection of cell fragments, debris and soluble cell fractions from patient specimens are found in large quantities in the blood or tissues of some cancer patients, allowing for MS analysis. The importance and potential diagnostic utility of cell debris detection has been the subject of pending U.S. application 10/780,399.
  • Figure 6 shows a diagramatic representation of one method for isolating the debri/cell fraction.
  • the components of the crude enriched whole blood fraction are separated by acidification to remove bovine serum ferrofluid (BSA-FF) and streptavidin, conjugated to a monoclonal antibody (streptavidin- Mab).
  • BSA-FF bovine serum ferrofluid
  • streptavidin- Mab conjugated to a monoclonal antibody
  • WBC White blood cells
  • Lipids, such as found in the membrane, are removed by solvent extraction.
  • the only remaining components are the rare cells of interest (i.e. tumor cells and/or endothelial cells) and serum protein/glycoproteins. These are enriched by N 2 evaporation.
  • the magnetically enriched fractions are retrieved from the viewing chamber after imaging by magnetic separation of the supernatant buffer and buffer components.
  • the buffer is replaced by an enzyme-compatible saline solution and analyzed directly.
  • reversible chemical dissociation or tryptic dissociation digestion into fragments prior to MS analysis are done within the chamber by adding a dissociating agent or enzyme solution to a suspension of the magnetic particles to separate the ferrofluid particles.
  • the captured cell and/or proteins are dissociated from the ferrofluid particles with an optional digestion to peptide fragments prior to analysis by MS.
  • the preferred size for MS detection after tryptic digestion is 7,000 to 20,000 Da. This is a range that is lower than the sizes of most soluble tumor markers, and much lower than the sizes reported for circulating tumor cell debris.
  • Another embodiment incorporates magnetic enrichment of the target cells and/or cell debris using a proteomic analysis system as the only platform. lmmunomagnetic enrichment provides a simple amplification method to improve the sensitivity to a level that allows for consistent diagnostic use.
  • the captured target cells or proteins, complexed with desthiobiotinylated monoclonal antibody-ferrofluid are assessed by MS either alone or in combination with image analysis.
  • the captured target cells or proteins are dissociated from the ferrofluid with biotin to generate and enriched sample fraction, free of proteins derived from the ferrofluid particles.
  • epithelial cell adhesion molecule (EpCAM) MAb-FF captures most of the target entities in the enriched sample fraction while other gradient methods may lose a substantial portion of entities.
  • Both approaches yield tumor specific mass profiles that are subtracted from MS profiles for BSA, MAbs-FF, or other sample enriched components. These subtracted profiles can be compared for disease and/or disease state, yet without knowledge of the identity of the measured proteins.
  • the two embodiments, mentioned above, allow for complementary confirmation of CTC obtained by imaging, or possibly earlier cancer diagnosis in MS analysis without associated imaging.
  • MS analysis can provide a means for early cancer diagnosis even before intact CTC are detectable by imaging from a small blood specimen.
  • Her2/neu levels in the low ng/ml range in plasma can be immunomagnetically enriched to provide debris levels sensitive enough for MS analysis.
  • MS proteomic analysis would obviate the need for immediate analysis or stabilization of blood samples for later analysis, required in image analysis.
  • controlled aggregation may be unnecessary when analyzing captured debris.
  • These factors could provide an improved sensitivity to diseases such as early cancer detection.
  • cell analysis platforms can be used to identify and enumerate cells in the enriched samples. Examples of such analytical platforms are described in US Patents 5,876,593; 5,985,153 and 6,136,182, each of which are incorporated by reference herein as disclosing the respective apparatus and methods for manual or automated quantitative and qualitative cell analysis.
  • 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);
  • 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.
  • CK-PE Phycoerythrin
  • 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 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 Pr0 , 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 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 image cytometry system consists of a microscope with a Mercury Arc Lamp, a 1OX 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 DiOCI 6 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 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 image 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 (DiOCi 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 4Dm, the presence of a nucleus surrounded by cytoplasmic cytokeratin staining and absence of DiOC-i 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 Dm 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 4mm 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 4mm 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 4mm with or without association of nuclear material that had no morphological resemblance to a cell.
  • CTC were enumerated in 18 blood samples of prostate cancer patients and 27 samples from healthy individuals by both flow cytometry and image cytometry The results shown in Table 1 were sorted by increasing number of intact CTC detected.
  • Table 1 Enumeration of CTC by image cytometry and flow cytometry in 18 blood samples of prostate cancer patients and 27 samples from healthy individuals.
  • FIGs 2G, 2H, and 21 the flow cytometric analysis of a blood sample spiked with 501 LnCaP cells is shown.
  • a predominantly bright cytokeratin positive population with a nucleic acid content greater than normal human leukocytes and relatively large size as illustrated by the large forward light scatter signals are shown and depicted black in the figure. Only few CK+, CD45- events with NAD content less than leukocytes and depicted blue are detected in the sample.
  • Figures 2J, 2K, and 2L shows the flow cytometric analysis of paclitaxel treated LnCaP cells spiked in blood.
  • CTC detected by both flow cytometry and image cytometry 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.
  • the 100Dl 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 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 100Dl assay.
  • the Suspect CTC are not created during sample processing (in vitro damage) as the 100DI 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.
  • CTC recoveries of CTC from some other clinical samples have been as low as 20%. There may be several factors that contribute for a lower recovery, such as EpCAM positive/cytokeratin negative cells, cytokeratin dim cells, and mucin on the cell surface inhibiting the ability of ferrofluid to bind cells.
  • Ratio Obvious CTC / Total CTC
  • Ratio Obvious CTC / Total CTC
  • the results are summarized in Table III. Ratios near 1.0 indicate the Total CTC are obvious CTC, and 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, and 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.
  • Table 4 shows that plasma washing eliminates at least 3 fold mRNA. Because intact CTCs do not remain in the plasma following centrifugation at
  • RNA signals must come from a fraction of cell debris that does not partition from the plasma fraction and remains in the plasma, subsequently aspirated away with washing. Consequently, an even larger difference could result with the incorporation of rare cell debris, partitioned from the plasma after centrifugation.
  • Intact CTC and genes expressed in epithelial cells can be detected in blood samples of CRC patients enriched for EpCAM expression.
  • the finding of RT-PCR positives in patients in which no intact CTC were detected may be due to carcinoma cells shed into the blood that have been damaged or destroyed.
  • 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 1-3Dm (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 percent 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.
  • Sequence analysis includes the quantification, and/or qualification of an individual sequence or groups of sequences associated with the disease of interest.
  • RNA sequence analysis is accomplished by multigene RNA profile analysis.
  • Sequence quantification is accomplished by quantitative RT-PCR while sequence qualification is accomplished through array analysis.
  • the present invention is not limited to this analysis, but includes all sequence analysis accepted by individuals in the field.
  • 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.

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Abstract

Les procédés et les réactifs décrits dans la présente invention permettent d’analyser les cellules tumorales en circulation, les grappes, les fragments et les débris. L’analyse est réalisée avec un certain nombre de plates-formes, y compris une cytométrie d’écoulement, l’imagerie par microscopie fluorescente CellSpotterTM et le profilage spectrométrique de masse. Outre l’analyse et l’énumération de cellules classées morphologiquement par imagerie, la présence des cellules endommagées et des débris dérivés dans des spécimens enrichis magnétiquement s’est révélée un indicateur important “ pistolet fumant ” de cellules tumorales tuées. L’invention décrit des procédés améliorés pour cribler, diagnostiquer et surveiller les maladies sur la base de cellules rares intactes en circulation et de fragments ou débris associés. La présente invention incorpore le profilage protéomique et génomique de débris cellulaires dans l’analyse des cellules cancéreuses, soit de manière complémentaire à ou indépendante de la cytométrie d’image. La technologie fournit des outils de diagnostic et de pronostic dans les maladies comme le cancer intégrant non seulement la détection et la quantification des cellules intactes en circulation mais également des fragments et débris cellulaires portant des marqueurs cytosquelettiques génériques et/ou spécifiques aux tumeurs. La présence de tels marqueurs reflète la fonction immunitaire au début de la maladie et/ou l’efficacité thérapeutique à des stades ultérieurs.
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US8609349B2 (en) 2008-02-25 2013-12-17 Nestec S.A. Drug selection for breast cancer therapy using antibody-based arrays
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US10401364B2 (en) 2011-02-03 2019-09-03 Soiété Des Produits Nestlé S.A. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
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