Classifications

• • 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
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Definitions

• the invention relates generally to cancer monitoring and assessing disease progression in metastatic cancer patients, based on the presence of morphologically intact circulating cancer cells (CTC) in blood. More specifically, methods, reagents and apparatus are described for assessing circulating cancer cells in animal models.
• CTC morphologically intact circulating cancer cells
• Non-hematogenous epithelial tumor cells were first identified in the blood of a breast cancer patient over 150 years ago. Since then, CTCs have been shown to be a critical link between primary cancer, a disease stage at which cure is possible, and metastatic disease, which continues to be the leading cause of death for most malignancies. Clinical studies have shown that CTCs are a powerful prognostic and predictive biomarker in metastatic breast cancer, and similar findings have been reported in prostate cancer and colorectal cancer. From this data, CTCs have been shown to be representative of the underlying biology driving metastatic cancer and suggest that further cellular and molecular analyses of these cells can reveal new insights into molecular regulation of metastasis and response to therapy.
• metastasis can be considered the conclusive event in the natural progression of cancer.
• the ability to metastasize is a property that uniquely characterizes a malignant tumor. Based on the complexity of cancer and cancer metastasis and the frustration in treating cancer patients over the years, many attempts have been made to develop diagnostic tests to guide treatment and monitor the effects of such treatment on metastasis or relapse.
• CEA carcinoembryonic antigen
• PSA levels in serum have proven to be useful in early detection.
• the PSA test has improved detection of prostate cancer at an early stage when it is best treated.
• PSA or the related PSMA testing leaves much to be desired.
• elevated levels of PSA weakly correlate with disease stage and appear not to be a reliable indicator of the metastatic potential of the tumor. This may be due in part to the fact that PSA is a component of normal prostate tissue and benign prostatic hyperplasia (BHP) tissue.
• BHP benign prostatic hyperplasia
• approximately serum PSA concentrations may have metastatic disease (Moreno et al., Cancer Research, 52:6110 (1992)).
• the test is performed on 10 5 to 10 6 cells separated from interfering red blood cells, corresponding to a practical lower limit of sensitivity of one tumor cell/0.1 ml of blood (about 10 tumor cells in one ml of blood) before a signal is detected.
• Higher sensitivity has been suggested by detecting hK2 RNA in tumor cells isolated from blood (US 6,479,263; US 6,235,486).
• Qualitative RT-PCR based studies with blood-based nucleotide markers has been used to indicate that the potential for disease-free survival for patients with positive CEA mRNA in pre-operative blood is worse than that of patients negative for CEA mRNA (Hardingham J. E., Hewett P.J., Sage R. E., Finch J.
• CEA mRNA levels which are also induced in healthy individuals by G-CSF, cytokines, steroids, or environmental factors.
• the CEA mRNA marker lacks specificity and is clearly not unique to circulating colorectal cancer cells.
• RT-PCR real-time reverse transcriptase-polymerase chain reaction
• telomeres were shed into the bloodstream (possibly during surgical procedures or from micro metastases already existing at the time of the operation), and resulted in poor patient outcomes in patients with colorectal cancer.
• the sensitivity of this assay provided a reproducibly detectable range similar in sensitivity to conventional RT-PCR. As mentioned, these detection ranges are based on unreliable conversions of amplified product to the number of tumor cells. The extrapolated cell count may include damaged CTC incapable of metastatic proliferation. Further, PCR-based assays are limited by possible sample contamination, along with an inability to quantify tumor cells.
• An alternative approach incorporates immunomagnetic separation technology and provides greater sensitivity and specificity in the unequivocal detection of intact circulating cancer cells.
• This simple and sensitive diagnostic tool as described (US6,365,362; US6.551.843; US6,623,982; US6,620,627; US6.645.731 ; WO 02/077604; WO03/065042; and WO 03/019141 ) is used in the present invention to provide a preclinical animal model to enumerate CTCs.
• the assay depends upon the acquisition of a preserved blood sample from a patient.
• the blood sample from a cancer patient (WO 03/018757) is incubated with magnetic beads, coated with antibodies directed against an epithelial cell surface antigen as for example EpCAM.
• the magnetically labeled cells are then isolated using a magnetic separator.
• the immunomagnetically enriched fraction is further processed for downstream immunocytochemical analysis or image cytometry, for example, in the CellTracks ® System (Veridex LLC, NJ).
• the magnetic fraction can also be used for downstream immunocytochemical analysis, RT-PCR, PCR, FISH, flowcytometry, or other types of image cytometry.
• the CellTracks® System utilizes immunomagnetic selection and separation to highly enrich and concentrate any epithelial cells present in whole blood samples.
• the captured cells are detectably labeled with a leukocyte specific marker and with one or more tumor cell specific fluorescent monoclonal antibodies to allow identification and enumeration of the captured CTCs as well as unequivocal instrumental or visual differentiation from contaminating non-target cells.
• This assay allows tumor cell detection even in the early stages of low tumor mass.
• the embodiment of the present invention is not limited to the CellTracks® System, but includes any isolation and imaging protocol of comparable sensitivity and specificity.
• the present invention provides a method and means for preclinical modeling of cancer metastasis in xenograft mice, incorporating clinical analysis tools such as the CellTracks® System, and is based upon the absolute number, change, or combinations of both of circulating epithelial cells in patients with metastatic cancer.
• the system immunomagnetically concentrates epithelial cells, fluorescently labels the cells, identifies and quantifies CTCs for positive enumeration in zenograft tumor models of human breast cancer.
• Figure 1 CellTracks® fluorescent analysis profile used to confirm objects captured as human tumor cells.
• Check marks signify a positive tumor cell based on the composite image.
• Composite images are derived from the positive selection for Epithelial Cell Marker (EC-PE) and for the nuclear dye (NADYE).
• EC-PE Epithelial Cell Marker
• NADYE nuclear dye
• a negative selection is also needed for the leukocyte marker (L- APC) and for control (CNTL).
• FIG. 2 Quantification of human breast cancer cells in mouse blood samples.
• MDA-MB-231 human breast cancer cells without or with stable transduction of GFP were added to 100 ⁇ l blood samples from mice without tumor xenografts.
• Samples were fixed, and epithelial cells were enriched by immunomagnetic bead isolation using an antibody to epithelial cell adhesion molecule.
• Recovered cells then were stained with an antibody to cytokeratin (8, 18, and 19) to identify epithelial cells and distinguish them from leukocytes stained with CD45.
• Nucleated cells were identified by staining with the fluorescent nucleic acid dye 4,2-diamidino-2-phenylindole dihydrochlohde (DAPI).
• DAPI fluorescent nucleic acid dye 4,2-diamidino-2-phenylindole dihydrochlohde
• Figure 3 Quantification of human breast cancer cells in mouse blood samples. Terminal blood samples from mice bearing xenografts of MDA-MB-231 human breast cancer cells were obtained by cardiac puncture and analyzed for CTC. Numbers of CTC are plotted versus tumor volumes measured by calipers.
• FIG. 4 Serial analysis of CTC in mice. Mice were implanted with orthotopic tumor xenografts of SUM-159 (A) or SKBR-3 (B) human breast cancer cells, and CTC in approximately 100 ⁇ l blood samples were measured by cardiac puncture at approximately weekly intervals until mice were euthanized because of tumor burden. CTC data were normalized to 100 ⁇ l volume and plotted against tumor volume for individual. Mean numbers of CTC were significantly greater on day 30 as compared with prior days (p ⁇ 0.05).
• 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 been shown to have 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 modeling disease progression in an animal model.
• Circulating tumor cells have been shown to exist in the blood in detectable amounts. This created a tool to investigate the significance of cells of epithelial origin in the peripheral circulation of cancer patients (Racila E., Euhus D., Weiss A.J., Rao C, McConnell J., Terstappen L.W.M.M. and Uhr J.W., Detection and characterization of carcinoma cells in the blood, Proc. Natl. Acad. Sci. USA, 95:4589-4594 (1998)). This study demonstrated that these blood-borne cells might have a significant role in the pathophysiology of cancer. Having a detection sensitivity of 1 epithelial cell per 5 ml of patient blood, the assay incorporated immunomagnetic sample enrichment and fluorescent monoclonal antibody staining followed by flowcytometry for a rapid and sensitive analysis of a sample.
• the CellSearchTM System (Veridex LLC, NJ) previously has been used to isolate and enumerate circulating epithelial tumor cells from human blood samples 2 .
• This is an automated system that enriches for epithelial cells using antibodies to epithelial-cell adhesion molecule coupled to magnetic beads. Isolated cells then are stained with the fluorescent nucleic acid dye 4,2- diamidino-2-phenylindole dihydrochlohde (DAPI) to identify nucleated cells. Recovered cells subsequently are stained with fluorescently labeled monoclonal antibodies to CD45 (APC channel) and cytokeratin 8, 18, 19 (PE channel) to distinguish epithelial cells from leukocytes. Nucleated epithelial cells then are quantified as circulating tumor cells. There is an additional fluorescence channel for FITC that is not part of the standard CellSearchTM assay and may be used for further characterization of tumor cells.
• the assay was further configured to an image cytometric analysis such that the immunomagnetically enriched sample is analyzed by the CellTracks® System.
• image cytometric analysis such that the immunomagnetically enriched sample is analyzed by the CellTracks® System.
• This is a fluorescence-based microscope image analysis system, which in contrast with flowcytomethc analysis permits the visualization of events and the assessment of morphologic features to further identify objects.
• the CellTracks® System refers to an automated fluorescence microscopic system 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 presents suspect cancer cells, labeled with antibodies to cytokeratin and having epithelial origin, to the operator for final selection..
• one embodiment uses immunomagentic enrichment for isolating tumor cells from a biological sample.
• 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 in the CellTracks® System.
• Cells are identified automatically in the CellTracks® System and candidate circulating tumor cells presented to the operator for checklist enumeration.
• An enumeration checklist consists of predetermined morphologic criteria constituting a complete cell.
• Cytokeratin positive cells are isolated by immunomagnetic enrichment using a 7.5 ml sample of whole blood from humans. 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 in the CellTracks® System.
• Cells are identified automatically in the CellTracks® System; control cells are enumerated by the system, whereas the candidate circulating tumor cells are presented to the operator for enumeration using a checklist as shown in Figure 1.
• MDA-MB-231 breast cancer cells were spiked into 100 ⁇ l blood samples collected from mice without tumors. Since the clinical version of the assay requires blood be drawn into a proprietary vacuum tube, such as the CellSave tube, containing both an anticoagulant and a preservative, a proportionately reduced amount of CellSave solution was added to the specimens. The spiked specimens were then prepared, the CTC quantified and the percent recovery calculated. As a positive control, additional samples using MDA-MB-231 cells stably transduced with GFP were prepared. Fluorescence from GFP was detected in an open channel (FITC) of the system to confirm that all cells quantified as epithelial cells corresponded with 231 -GFP cells added to mouse blood.
• FITC open channel
• the preferred method to serially monitor CTCs in mouse models of human breast cancer incorporates the use of the CellTracks® System.
• the system uses immunomagnetic isolation of epithelial cells from blood and immunofluorescent staining to further differentiate epithelial cancer cells from leukocytes. Because the CellTracks® system was originally developed to process 7.5 to 30 ml human blood samples, it is necessary that human epithelial breast cancer cells could be reliably recovered from small volumes of mouse blood using this assay (see Example 2).
• the system was used to identify CTCs that spontaneously intravasate into the circulation from orthotopic tumor xenografts of MDA-MB-231 cells.
• 0.7 to 1 ml blood samples were collected from each mouse by puncture of the left ventricle when animals were euthanized for tumor burden at 10 weeks.
• Total numbers of CTCs ranged from approximately 100 to 1000 cells per ml of blood ( Figure 3).
• No CTCs were recovered from blood samples collected from mice without tumor xenografts (data not shown). The number of CTCs did not correlate with size of the primary tumor.
• CTCs were also detectable in mice with tumor xenografts of MCF-7, MCF-7 cells stably transfected with fibroblast growth factor (FGF), SUM-159, and SKBR-3 cell-lines. While the system was successful in detecting CTCs using cardiac puncture to collect blood, this procedure is invasive compared to other sites of blood sampling in mice.
• One aspect of the present invention is to repetitively draw blood samples for analysis of CTCs, blood samples from the lateral tail vein and retro-orbital venous plexus and thereby avoid the invasive nature of cardiac puncture. In mice with or without orthotopic MDA-MB-231 tumor xenografts were compared to direct cardiac sampling.
• MDA-MB-231 and SKBR-3 human breast cancer cells were cultured in DMEM with 10% fetal bovine serum, 1 % L-glutamine, and 0.1 % penicillin/streptomycin.
• SUM-159 cells were cultured in Ham's F12 medium (Invitrogen) supplemented with 5% fetal bovine serum (FBS), 5 ⁇ g/ml insulin, 1 ⁇ g/ml hydrocortisone, and 0.1 % penicillin/streptomycin. Cells were maintained at 37 0 C in a 5% CO2 incubator. For selected experiments, MDA-MB-231 cells were transduced with the lentiviral vector pSico to establish cells that stably express GFP. Efficiency of transduction was 100%, as determined by phase-contrast and fluorescence microscopy.
• mice In producing tumor xenografts in mice, 5 to 6 week old female Ncr nude (Taconic) or SCID (Jackson) mice were used.
• Human breast tumor xenografts from cell lines 1 x 10 6 cells were injected orthotopically into bilateral inguinal mammary fat pads of mice by methods know in the art.
• mice For tumor xenografts with clinical isolates of human breast cancer cells, mice were implanted with 1 - 5 x 10 5 cells in the fourth inguinal mammary fat pad. Mice implanted with clinical breast cancer isolates also received a subcutaneous pellet of 60-day sustained release17- ⁇ -estradiol (Innovative Research of America).

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