WO2006041453A1 - Cellules tumorales circulantes (ctc): evaluation de l'apoptose chez les patients presentant un cancer de la prostate - Google Patents

Cellules tumorales circulantes (ctc): evaluation de l'apoptose chez les patients presentant un cancer de la prostate Download PDF

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WO2006041453A1
WO2006041453A1 PCT/US2004/032029 US2004032029W WO2006041453A1 WO 2006041453 A1 WO2006041453 A1 WO 2006041453A1 US 2004032029 W US2004032029 W US 2004032029W WO 2006041453 A1 WO2006041453 A1 WO 2006041453A1
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cells
cancer
cell
apoptotic
sample
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PCT/US2004/032029
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Christopher J. Larson
Leon W. M. M. Terstappen
Jose G. Moreno
Kenneth J. Pienta
Madeline L. Repollet
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Immunivest Corporation
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Publication of WO2006041453A1 publication Critical patent/WO2006041453A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • CTCs Circulating Tumor Cells
  • This invention relates generally to the field of diagnostic testing, and more particularly to diagnostics in the oncology field.
  • the invention is useful in cancer screening, staging, monitoring for chemotherapy treatment responses, cancer recurrence or the like. More specifically, the present invention provides reagents, methods and test kits that facilitate analysis and enumeration of tumor cells, or other rare cells isolated from biological samples.
  • the invention also provides materials and methods for assessing tumor diathesis associated molecules, such as nucleic acids, proteins and carbohydrates, thereby aiding the clinician in the design of therapeutic treatment strategies.
  • metastases multiple widespread tumor colonies established by malignant cells that detach themselves from the original tumor and travel through the body, often to distant sites. If a primary tumor is detected at an early stage, it can often be eliminated by surgery, radiation, or chemotherapy or some combination of these treatments. Unfortunately, metastatic colonies are frequently more difficult to detect and eliminate and it is often impossible to treat all of them successfully. Therefore, from a clinical point of view, metastasis can be considered the penultimate event in the natural progression of cancer. Moreover, the ability to metastasize is the property that uniquely characterizes a malignant tumor.
  • Cancer metastasis comprises a complex series of sequential events. These are: 1) extension from the primary locus into surrounding tissues; 2) penetration into body cavities and vessels; 3) release of tumor cells for transport through the circulatory system to distant sites; 4) reinvasion of tissue at the site of arrest; and 5) adaptation to the new environment so as to promote tumor cell survival, vascularization and tumor growth.
  • PSA Prostate Specific Antigen
  • a useful diagnostic test needs to be very sensitive and reliably quantitative. If a blood test can be developed where the presence of a single tumor cell can be detected in 1 ml of blood, that would correspond on average to 3000-4000 total cells in circulation, lnnoculum studies for establishing tumors in animals show that injection of 3000-4000 of cells can indeed lead to the establishment of a tumor. Further if 3000-4000 circulating cells represent 0.01 % of the total cells in a tumor, then it would contain about 4 x 10 7 total cells. A tumor containing that number of cells would not be visible by any technique currently in existence. Hence, if tumor cells are shed in the early stages of cancer, a test with the sensitivity mentioned above would detect the cancer.
  • Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, US Patent No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinburgh (1983). Generally, any material that facilitates magnetic or gravitational separation may be employed for this purpose. However, it has become clear that 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.
  • Small 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.
  • a system with 0.8% NSB that recovers 80% of a population which is at 0.25% in the original mixture will have a purity of 25%.
  • the initial population was at 0.01% (one target cell in 10 6 bystander cells), and the NSB were 0.001%, then the purity would be 8%.
  • the purity of the target material in the specimen mixture results in a more specific and effective collection of the target material.
  • Extremely low non-specific binding is required or advantageous to facilitate detection and analysis of rare cells, such as epithelial derived tumor cells present in the circulation.
  • high gradient magnetic separation with an external field device employing highly magnetic, low non-specific binding, colloidal magnetic particles is the method of choice for separating a cell subset of interest from a mixed population of eukaryotic cells, particularly if the subset of interest comprises but a small fraction of the entire population.
  • Such materials because of their diffusive properties, readily find and magnetically label rare events, such as tumor cells in blood.
  • the magnetic particles must be specific for epitopes that are not present on hematopoeitic cells.
  • a method for detecting and enumerating apoptotic cancer cells in a mixed cell population by obtaining a biological specimen, the presence of said cancer cells in said population being indicative of a disease state comprises: a. obtaining a biological specimen from a test subject, said specimen • comprising a mixed cell population suspected of containing said cancer cells; b. preparing an immunomagnetic sample wherein said specimen is mixed with magnetic particles coupled to a biospecific ligand which reacts specifically with said cancer cells, to the substantial exclusion of other sample components; c. contacting said immunomagnetic sample with at least one biospecific reagent which labels said cancer cells; and d.
  • a method for assessing a patient for the presence of a malignancy entails obtaining a mixed cell population suspected of containing hematopoietic and non-hematopoietic malignant cells, immediately collected in preservative tubes, containing a stabilizing agent and fixative agent. After enrichment, the sample is then prepared wherein the biological sample is mixed with a detectably labeled ligand which reacts specifically with the malignant cells, to the substantial exclusion of other sample components.
  • the sample is then contacted with at least one reagent which also specifically labels said malignant cells in an apoptotic state.
  • One embodiment of the present invention uses the M30-FITC antibody to detect the apoptotic state. This antibody is specific for apoptosis-induced caspase cleaved cytokeratin 18. Analysis of the sample is then performed to determine the presence and number of labeled cells, detection of said cells indicating the presence of malignancy. Either the greater the number of labeled intact cells present, the greater the number of apoptotic cells present, or a combination of both is suggestive of the severity of the malignancy.
  • cells are analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated image analysis.
  • kits are provided for screening a patient sample for the presence of non-hematopoietic malignant cells.
  • Figure 1 lmmunomagnetic Enrichment of whole blood sample prior to CTC analysis.
  • Semi-automated fluorescent analysis used CD45-APC, Cytokeratin (CK) PE, and DAPI as staining reagents.
  • Flow cytometry analysis used CD45-PerCP, CK PE, and nucleic acid dye as staining reagents.
  • Figure 2 Analysis by fluorescent microscopy CTC analysis from a 7.5 ml blood sample from a metastatic cancer patient. The columns of thumbnails correspond to the images from corresponding fields of the nucleus (DAPI), cytokeratin (CK-PE), control, leukocyte (CD45 APC) and false color overlay image of cytokeratin (green) and the nucleus (magenta).
  • the rows of numbered thumbnails present the different locations in the analysis cartridge that contain CTC candidates.
  • the center box of the "cross-hairs" in the lower right corner is 4 ⁇ m and is used to size individual events during review of the images ensuring a minimal cell size of 4 ⁇ m.
  • Figure 3 Flow cytometric CTC analysis of a sample previously analyzed by microscopy. Panel A and B shows all events whereas in Panels C and D only the cytokeratin positive events are shown.
  • Figure 4 Fluorescent microscopy and flow cytometric CTC analysis in three prostate cancer patients.
  • 53 of 240 intact CTC 7 of 20 damaged and 19 of 238 fragments are shown in Panel A.
  • Green represents cytokeratin and magenta the nucleus.
  • the corresponding flow cytometric analysis of only the cytokeratin positive events is shown in Panel B.
  • 2 of 16 intact CTC 33 of 40 damaged and 6 of 320 fragments are shown in Panel C and the corresponding flow cytometric analysis is shown in Panel D.
  • Panel E For patient five, 2 of 16 intact CTC, 15 of 38 damaged and 49 of 490 fragments are shown in Panel E and the corresponding flow cytometric analysis is shown in Panel F.
  • Figure 5 Fluorescent microscopy and flow cytometric CTC analysis of LnCaP cells spiked in blood of normal donors.
  • Panel A shows the images of untreated LnCaP cells and Panel B the corresponding flow cytometric analysis of only the cytokeratin positive events.
  • Panel C shows the images of paclitaxel treated LnCaP cells and Panel D the corresponding flow cytometric analysis.
  • tumor cells are identified in circulation, it is desirable to further characterize the isolated cells phenotypically or biochemically.
  • particular tumor diathesis associated molecules such as nucleic acid molecules, proteins, or carbohydrates that are associated with the malignant phenotype may be analyzed.
  • methods are provided in accordance with the invention for measuring the level of expression of predetermined tumor diathesis associated molecules present in or on tumor cells identified in the circulation in a blood sample where these molecules have been morphologically and antigenically preserved.
  • Classification of the apoptotic state of the tumor cells provides strict criteria in defining an intact CTC and their possible invasive potential. Further, classification and enumeration of apoptotic tumor cells provides critical information about the disease state. This information assists the clinician in diagnosing the type of cancer and assessing the efficacy of chemotherapeutic intervention strategies.
  • Another object of this invention is the improved detection of circulating tumor cells as an early prognostic indicator of a patient's disease state.
  • the method of the invention can be used to assess residual cancer cells in the circulation following medical radiation or surgical treatment to eradicate the tumor.
  • the method can also be performed periodically over the course of years to assess the patient for the presence and number or tumor cells in the circulation, and alterations in tumor diathesis molecules therein as an indicator of occurrence, recurrence and/or progression of disease as previously described (PCT/US04/05848, PCT/US99/03073, and U.S. 6,365,362)
  • An exemplary method comprises obtaining a sample from a patient; isolating and enumerating circulating malignant cells from said sample if present, and determining the number of at least one predetermined tumor diathesis associated molecule associated with apoptosis on individual cells present in said sample as a means to predict efficacy of therapy.
  • Such methods may also be used to an advantage to assess the appropriate dosage of a given therapeutic regimen and/or for monitoring the efficacy of therapy over time.
  • methods for culturing tumor cells isolated from the circulation are provided. Such cells may then be contacted with therapeutic agents to assess their sensitivity thereto. Such cells also provide a source for tumor diathesis associated molecules which may or may not be altered.
  • An exemplary kit of the invention comprises coated magnetic nanoparticles comprising: i) a preservative ii) a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said malignant cell, the antibody being coupled, directly or indirectly, to said base coating material; iii) at least one antibody having binding specificity for a second characteristic determinant of said malignant cell; iv) a cell specific dye for excluding sample components other than said malignant cells from analysis; v) a sample cartridge used in imaging; and at least one detectably labeled agent recognizing the cleavage of cytokeratin 18 after caspase cleavage.
  • Such kits may optionally comprise an antibody which has binding affinity for non-target cells, a biological buffer, a permeabilization buffer, a protocol and optionally, an information sheet.
  • one method for collecting circulating tumor cells combines immunomagnetic enrichment technology, immunofluorescent labeling technology with an appropriate analytical platform after initial blood draw.
  • the associated test has the sensitivity and specificity to detect these rare cells in a sample of whole blood and to investigate their role in the clinical course of the disease in malignant tumors of epithelial origin. From a sample of whole blood, rare cells are detected with a sensitivity and specificity to allow them to be collected and used in the diagnostic assays of the invention, namely predicting the clinical course of disease in malignant tumors.
  • CTC circulating tumor cells
  • the assay incorporates immunomagnetic sample enrichment and fluorescent monoclonal antibody staining followed by flowcytometry for a rapid and sensitive analysis of a sample.
  • the results show that the number of epithelial cells in peripheral blood of eight patients treated for metastatic carcinoma of the breast correlated with disease progression and response to therapy. In 13 of 14 patients with localized disease, 5 of 5 patients with lymph node involvement and 11 of 11 patients with distant metastasis, epithelial cells were found in peripheral blood. The number of epithelial cells was significantly larger in patients with extensive disease.
  • the assay was further configured to an image cytometric analysis such that the immunomagnetically enriched sample is analyzed by an automated imaging system.
  • image cytometric analysis such that the immunomagnetically enriched sample is analyzed by an automated imaging system.
  • This is a fluorescence-based microscope image analysis system, which in contrast with flowcytometric analysis permits the visualization of events and the assessment of morphologic features to further identify objects.
  • Automated fluorescence imaging provides for automated enumeration of isolated cells from blood.
  • the system contains an integrated computer controlled fluorescence microscope and automated stage with a magnetic yoke assembly that will hold a disposable sample cartridge.
  • the magnetic yoke is designed to enable ferrofluid-labeled candidate tumor cells within the sample chamber to be magnetically localized to the upper viewing surface of the sample cartridge for microscopic viewing.
  • Software detects cancer cells, labeled with an antibody of cytokeratin and having epithelial origin, from blood.
  • one embodiment uses a stabilizing and fixative process for isolating tumor cells from 7.5 ml of whole blood.
  • Epithelial cell-specific magnetic particles are added and incubated for 20 minutes. After magnetic separation, the cells bound to the immunomagnetic-linked antibodies are magnetically held at the wall of the tube. Unbound sample is then aspirated and an isotonic solution is added to resuspend the sample.
  • a nucleic acid dye, monoclonal antibodies to cytokeratin (a marker of epithelial cells) and CD 45 (a broad-spectrum leukocyte marker) are incubated with the sample.
  • the unbound fraction is again aspirated and the bound and labeled cells are resuspended in 0.2 ml of an isotonic solution.
  • the sample is suspended in a cell presentation chamber and placed in a magnetic device whose field orients the magnetically labeled cells for fluorescence microscopic examination.
  • Cells are identified automatically with control cells enumerated and candidate circulating tumor cells presented to the operator for checklist enumeration.
  • An enumeration checklist consists of predetermined morphologic criteria constituting a complete cell.
  • method and kits are provided for the rapid enumeration and characterization of tumor cells shed into the blood in metastatic and primary patients which can readily be assessed for individual cell integrety, based upon apoptotic state. This analysis has potential for prognostic assessment in survival and progression-free survival predictions.
  • the methods of the invention are useful in assessing a favorable or unfavorable survival, and even preventing unnecessary therapy that could result in harmful side-effects when the prognosis is favorable.
  • the present invention can be used for prognosis of any of a wide variety of cancers, including without limitation, solid tumors and leukemia's including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (i.e.
  • neoplasms i.e. bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital, neurofibromatosis, and cervical dysplasia.
  • Cytokeratin positive cells are isolated by stabilizing and fixing the mix population in a 7.5 ml sample of whole blood. Epithelial cell-specific immunomagnetic fluid is added and incubated for 20 minutes. After magnetic separation for 20 minutes, the cells bound to the immunomagnetic-linked antibodies are magnetically held at the wall of the tube. Unbound sample is then aspirated and an isotonic solution is added to resuspend the sample. A nucleic acid dye, monoclonal antibodies to cytokeratin (a marker of epithelial cells) and CD 45 (a broad-spectrum leukocyte marker) are incubated with the sample for 15 minutes.
  • the unbound fraction is again aspirated and the bound and labeled cells are resuspended in 0.2 ml of an isotonic solution.
  • the sample is suspended in a cell presentation chamber and placed in a magnetic device whose field orients the magnetically labeled cells for fluorescence microscopic examination by automated image analysis. Cells are identified automatically; control cells are enumerated, and the candidate circulating tumor cells are presented to the operator for enumeration by fluorescent microscopy ( Figure 1).
  • Figure 2 depicts a typical characterization of CTC by fluorescent microscopy of a blood sample from a patient with metastatic prostate cancer. Regions that potentially contain CTC are displayed in rows of thumbnails. The ruler in the left lower corner of the figure indicates the size of the thumbnails. From right to left these thumbnails represent nuclear (DAPI), cytoplasmic cytokeratin (CK-PE), control (no reagent) and surface CD45 (CD45-APC) staining. The composite images are displayed a false color overlay of nuclear (DAPI) and cytoplasmic (CK-PE) staining.
  • DAPI nuclear
  • CK-PE cytoplasmic cytokeratin
  • CD45-APC surface CD45
  • the check box beside the composite image allows the user to confirm that the images represented in the row are consistent with a tumor cell and the check box beside the CD45-APC image is used to identify a leukocyte or tumor cell that stained non-specifically.
  • 2761 rows of thumbnails were detected by the software that demonstrated staining consistent with tumor cells.
  • 12 are shown in Figure 2 and labeled 1632-1638 and 1869-1873.
  • Rows numbered 1636, 1638 and 1873 are checked off and display features of intact CTC defined as a size greater than 4 ⁇ m, the presence of a nucleus surrounded by cytoplasmic cytokeratin staining and absence of control and CD45 staining.
  • the cell in row 1638 is large and the one in row 1636 is significantly smaller.
  • the immunophenotype of the event in row 1869 is consistent with a tumor cell but the morphology shows a large nucleus with speckled cytoplasm due to retraction of cytoskeletal proteins.
  • 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. In this instance, the morphology of the cell closely resembles that of a lymphocyte.
  • thumbnails shown in rows 1633, 1635, 1637, 1870 and 1872 shows cytokeratin staining objects that are larger that 4 ⁇ m but have no resemblance to cells.
  • the cytokeratin positive objects in thumbnails 1637 and 1872 are in close proximity of a leukocyte. Based on the observation of images of CTC candidates in several patient samples, CTC were classified into three categories, intact CTC, damaged CTC and CTC fragments none of which stained with CD45 nor appeared in the control channel.
  • Figure 3 shows an example of the flow cytometric analysis of a blood sample from a patient with metastatic prostate cancer that was previously analyzed by fluorescent microscopy.
  • Panel A shows the CD45-APC and CK- PE staining of all events in the sample and
  • Panel B the DAPI and CK-PE staining.
  • the darker events that stain with CD45-APC and have a normal DNA content as evidenced by their presence in the lower right hand quadrant of Panel B are intact leukocytes.
  • the events that are positive for cytokeratin and are present in the upper right quadrant of Panel B are CTC with normal DNA content.
  • the events that are positive for cytokeratin are CTC with insufficient DNA content, shown in the upper left quadrant of Panel B. All other events are depicted gray.
  • Panels C and D are from the same experiments but only the cytokeratin positive events are shown.
  • the CK-PE positive and CD45 APC negative events were subdivided based on staining with M30 FITC and DAPI, as shown in Panel D.
  • Events that were positive for DAPI and negative for M30 are shown in the upper left portion of Panel C and the lower right quadrant of Panel D.
  • Events that were positive for DAPI and M30 are shown in the upper right quadrant of Panel D.
  • Events that were negative for DAPI and positive for M30 are shown in upper left quadrant of Panel D.
  • Events that were negative for DAPI and M30 are shown in the lower left quadrant of panel D.
  • the arrow in Panel D indicates the apoptotic pathway of CTC.
  • Figure 4 exemplifies the analysis of CTC by fluorescent microscopy and flow cytometry from blood samples. Individual thumbnails from the CTC images are shown as a composite and divided into three morphological distinct categories separated by arrows from left to right, intact CTC, damaged CTC and CTC fragments.
  • the flow cytometry plot of DAPI versus M30 FITC staining of the cytokeratin positive events of the same sample is shown to the right of the collection of images.
  • the number and relative frequency of the DAPI pos ,M30 neg ; DAPI po Mvl30 pos ; DAPI dim , M30 pos and DAPI dim , M30 neg events are provided in the figure.
  • CTC In the blood of patient 10 shown in Panel A, the majority of CTC are intact and 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 cytoplasm. This population most likely correlates with the DAPI pos , M30 neg and DAPI pos , M30 pos regions on the corresponding flow plot in Panel B. Fewer CTC were identified as damaged or cell fragments.
  • Table 1 summarizes the findings obtained by microscopy and flow cytometry of each of the 10 patients.
  • the analysis of blood samples from healthy men is shown in the table.
  • 0 events classify as intact CTC by microscopy and no more than two were detected by flow cytometry.
  • few events were found that classified as damaged or CTC fragments.
  • R 2 0.95.
  • the paclitaxel treated cells displayed speckled cytokeratin staining and irregular morphology.
  • the predominant population showed a decreased in DAPI staining (87%) and 26% of these cells express M30.
  • the apoptotic pattern of CTC in the patient samples closely resembles that of the paclitaxel treated LnCaP cells.

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Abstract

L'invention concerne un procédé permettant de détecter et de dénombrer les cellules cancéreuses apoptotiques dans une population cellulaire mixte au moyen d'un prélèvement, la présence desdites cellules cancéreuses dans ladite population signalant un état pathologique. Ce procédé consiste opérer un prélèvement biologique chez un sujet examiné, ledit prélèvement contenant une population cellulaire mixte suspectée de contenir lesdites cellules cancéreuses, à préparer un échantillon immunomagnétique en mélangeant ledit prélèvement avec des particules magnétique couplées à un ligand biospécifique réagissant spécifiquement avec lesdites cellules cancéreuses sensiblement à l'exclusion des autres composants de l'échantillon, à mettre en contact ledit échantillon immunomagnétique avec au moins un réactif biospécifique permettant de marquer lesdites cellules cancéreuses, et à caractériser lesdites cellules cancéreuses selon un ou plusieurs états apoptotiques, au moyen d'une analyse choisie dans le groupe comprenant l'analyse enzymatique, l'analyse biochimique, l'analyse morphologique, ou de combinaisons de celles-ci, la présence et le nombre desdits états apoptotiques étant indicatifs dudit état pathologique.
PCT/US2004/032029 2004-09-30 2004-09-30 Cellules tumorales circulantes (ctc): evaluation de l'apoptose chez les patients presentant un cancer de la prostate WO2006041453A1 (fr)

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US8986944B2 (en) 2001-10-11 2015-03-24 Aviva Biosciences Corporation Methods and compositions for separating rare cells from fluid samples
US8980568B2 (en) 2001-10-11 2015-03-17 Aviva Biosciences Corporation Methods and compositions for detecting non-hematopoietic cells from a blood sample
US8969021B2 (en) 2001-10-11 2015-03-03 Aviva Biosciences Corporation Methods and compositions for detecting non-hematopoietic cells from a blood sample
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US10613089B2 (en) 2006-01-30 2020-04-07 The Scripps Research Institute Method of using non-rare cells to detect rare cells
US8940493B2 (en) 2006-06-02 2015-01-27 Veridex Llc Circulating tumor cell assay
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US10155984B2 (en) 2006-06-14 2018-12-18 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags
US10591391B2 (en) 2006-06-14 2020-03-17 Verinata Health, Inc. Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US8372584B2 (en) 2006-06-14 2013-02-12 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags
US9347100B2 (en) 2006-06-14 2016-05-24 Gpb Scientific, Llc Rare cell analysis using sample splitting and DNA tags
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US9267943B2 (en) 2007-12-12 2016-02-23 The Board Of Trustees Of The Leland Stanford Junior University Apparatus for magnetic separation of cells
US8071395B2 (en) 2007-12-12 2011-12-06 The Board Of Trustees Of The Leland Stanford Junior University Methods and apparatus for magnetic separation of cells
WO2009089254A1 (fr) * 2008-01-07 2009-07-16 Luminex Corporation Capture et imagerie immunomagnétiques de cibles biologiques
US8372590B2 (en) 2008-01-07 2013-02-12 Luminex Corporation Isolation and enumeration of cells from a complex sample matrix
US8318445B2 (en) 2008-01-07 2012-11-27 Luminex Corporation Immunomagnetic capture and imaging of biological targets
US8195415B2 (en) 2008-09-20 2012-06-05 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnosis of fetal aneuploidy by sequencing
US9404157B2 (en) 2008-09-20 2016-08-02 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnosis of fetal aneuploidy by sequencing
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US10669585B2 (en) 2008-09-20 2020-06-02 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnosis of fetal aneuploidy by sequencing
US8296076B2 (en) 2008-09-20 2012-10-23 The Board Of Trustees Of The Leland Stanford Junior University Noninvasive diagnosis of fetal aneuoploidy by sequencing
US10527624B2 (en) 2014-01-27 2020-01-07 Epic Sciences, Inc. Circulating tumor cell diagnostics for prostate cancer biomarkers
US10545151B2 (en) 2014-02-21 2020-01-28 Epic Sciences, Inc. Methods for analyzing rare circulating cells
US11340228B2 (en) 2014-02-21 2022-05-24 Epic Sciences, Inc. Methods for analyzing rare circulating cells
WO2017196044A3 (fr) * 2016-05-09 2018-08-02 주식회사 싸이토젠 Système et procédé de criblage d'agent anticancéreux personnalisé pour un patient présentant une surexpression du récepteur des androgènes en utilisant les cellules tumorales circulantes du sang du patient atteint d'un cancer de la prostate
WO2023159427A1 (fr) * 2022-02-24 2023-08-31 青岛华赛伯曼医学细胞生物有限公司 Procédé de détection et d'isolement de la population cellulaire co-exprimant cd45 et epcam et son utilisation

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