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
• • 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
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
  • B01L3/5088—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above confining liquids at a location by surface tension, e.g. virtual wells on plates, wires
• • 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
  • G01N33/483—Physical analysis of biological material
  • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
• • 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
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • 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
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/365—Control or image processing arrangements for digital or video microscopes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0822—Slides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/16—Surface properties and coatings
  • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic

Definitions

• the invention relates generally to medical diagnostics and more specifically to identifying and categorizing circulating tumor cells (CTCs).
• CTCs circulating tumor cells
• CTCs circulating tumor cells
• healthy donor blood is generally used as a negative control, and while published numbers vary, in general only low numbers of CTCs are found in clinically healthy people.
• the healthy donor population consists of non- laboratory member volunteers of varying ages who are not known to have cancer but who have not undergone extensive medical evaluations for occult cancers. Since all cancer is, of course, initially asymptomatic, finding a truly negative population for specificity
• FIG. 1 shows a plan view of a three well plate as used in prior art static imaging systems.
• a slide includes three equally sized well regions.
• each well region 10 is a 1.45 cm square.
• the resulting total well area for the slide is 6.3 cm .
• the periphery of the wells is 17.4 cm.
• the percentage of the overall slide occupied by the three wells is 23.6%.
• An estimated number of cells per slide is in the range from 1.25 to 1.5 million cells.
• FIG. 2 shows a plan view of a twelve well plate as used in the prior art.
• a slide includes twelve equally sized well regions 12.
• each well region is a circle with a 0.5 cm diameter.
• the resulting total well area for the slide is 2.4 cm .
• the periphery of the wells is 19 cm.
• the percentage of the overall slide occupied by the three wells is 12.8%.
• An estimated number of cells per slide is in the range from 500 to 600 thousand cells.
• Apparatus, system and method are provided for the identification of various objects, particularly CTCs.
• the invention provides a system for assaying cells.
• the system includes a well of the present invention, an illumination system, an imaging system, an analysis module having functionality for analyzing cell selection criteria, and a user output.
• system may include, but is not limited to, a scanning system, an image storage system, and an analysis system
• the analysis system preferably identifies desired objects, such as complete cells, based on various criteria, which may include cell nuclear area or volume, CD-45 negative status, and cytokeratine positive status.
• a slide bearing a well for containing the cells during the imaging step, the well including a planar bottom surface, a border at the periphery of the well defining sides for the well, the border being adjacent the bottom surface of the well and providing a fluidic seal there between.
• the invention provides a well for assaying cells which is disposed on a surface of a substrate.
• the well includes a planar bottom surface, and a border forming a periphery of the well, the border being adjacent the bottom surface and providing a fluidic seal therebetween.
• Embodiments of the invention provide for a single imaging well, providing for substantially a monolayer of objects, e.g., cells.
• the well has an area preferably greater than 7.5 cm 2 , more preferably greater than 10 cm 2 , and most preferably substantially 11.7 cm .
• the perimeter of the well is preferably substantially 12.5 cm, more preferably substantially 14.5 cm and most preferably substantially 15.7cm, correspondingly.
• the percentage of the top surface of the slide covered by the well is preferably substantially 40%, more preferably 53% and most preferably substantially 62%.
• the wells are sized so as to permit the imaging of a monolayer of preferably 1.6 to 1.9 million cells, more preferably 2.1 to 2.6 million cells, and most preferably, 2.5 to 3 million cells, respectively.
• the preferred imaging wells have a total of four sides. By reducing the number of sides (as compared to the prior art, for example, the 3 well slide has 12 sides) and their perimeter, the edge effects associated with the side wall boundary are minimized.
• the approach used herein to identify CTCs in high definition (HD-CTCs) is distinct in that it does not rely on any single protein enrichment strategies. Instead, all nucleated cells are retained and immunofluorescently stained with monoclonal antibodies targeting cytokeratin (CK), an intermediate filament found exclusively in epithelial cells, a pan leukocyte specific antibody targeting CD45, and a nuclear stain, DAPI.
• CK cytokeratin
• the nucleated blood cells are imaged in multiple fluorescent channels to produce high quality and high resolution digital images that retain fine cytologic details of nuclear contour and cytoplasmic distribution.
• This enrichment-free strategy results in high sensitivity and high specificity, while adding high definition cytomorphology to enable detailed morphologic characterization of a CTC population known to be heterogeneous.
• An advantage of this approach is that multiple analysis parameters can be pursued to identify and characterize specific populations of interest.
• Embodiments of the instant inventions have been used to assay samples using "HD-CTCs" in metastatic cancer patients.
• the key innovative aspects of this assay are its simplicity, with minimal processing to the blood specimen, and its ability to enable professional morphologic interpretation with diagnostic pathology/cytopathology quality imagery.
• the invention provides a method for performing a cellular assay.
• the method includes contacting a sample having a population of cells with the well of the present invention, and analyzing the population of cells via the system of the present invention, thereby preforming the cellular assay.
• the analysis includes characterization of cell types within the population of cells, such as CTCs.
• the invention provides a method of detecting a CTC in a sample.
• the method includes contacting the well of the present invention with the sample, analyzing the population of cells via the system of the present invention; and detecting a CTC based on the analysis, thereby detecting a CTC in the sample.
• more than 2, 5, 7, 10, 15, 20 or 50 circulating tumor cells are detected per ml of sample.
• the invention provides a method for diagnosing cancer or providing a prognosis for cancer in a subject.
• the method includes contacting a well of the present invention with a sample including a population of cells from the subject, analyzing the population of cells via the system of the present invention, detecting a CTC in the cell population, characterizing the CTC, and determining a diagnosis or prognosis based on the characterization, thereby diagnosing or providing a prognosis for cancer in the subject.
• the invention provides a method for determining
• the method includes contacting the well of the present invention with a sample including a population of cells from a subject, analyzing the population of cells via the system of the present invention, detecting a CTC via the analysis, and characterizing the CTC to determine efficacy of administration of a chemotherapeutic agent, thereby determining responsiveness of the subject to the therapeutic regime.
• the invention provides a kit.
• the kit includes at least one well of the present invention, reagents for immunologically determining the presence of cytokeratin or CD45 in a cell, and instructions for utilizing the kit the detect a CTC in a sample.
• HD-CTC preferably requires one or more of the requirements that the cell(s) have an intact nucleus, express cytokeratin and not CD45, be morphologically distinct from surrounding benign white blood cells (WBCs), and have cytologic features consistent with intact morphologically abnormal epithelial cells suitable for downstream analysis.
• WBCs benign white blood cells
• FIG. 1 is a plan view of a prior art 3 well plate.
• FIG. 2 is a plan view of a prior art 12 well plate.
• FIG. 3 is a functional block diagram of the overall HD CTC system.
• FIG. 4 is a component level diagram of the scanning and imaging components of the system.
• FIG. 5 shows possible image slices through the measured parameter space, representing filtering of cell based on these parameters.
• FIG. 6 is a plan view of a representative slide and well.
• FIG. 7 is a perspective view of a representative slide and well.
• FIG. 8 shows the mean observed SKBR3s plotted against expected SKBR3s.
• FIG. 9 shows a comparison of CTC counts between two separate processors on 9 different cancer patient samples.
• CTC/mL counts ranged from 0 to 203.
• FIG. 10 shows comparative test data of the systems, apparatus and methods described here, versus the CellSearch® product.
• FIG. 11 shows test results graphing the quantity of CTCs for various samples, for prostate, pancreatic, breast tumors, and a comparison to healthy population.
• FIG. 12 shows the quantity of CTCs for various patient samples relative to breast cancer.
• FIG. 13 shows the normalized nuclear area versus nuclear area for white blood cells (WBCs) and CTCs, including a blow-up of the base-line region.
• WBCs white blood cells
• CTCs including a blow-up of the base-line region.
• a circulating tumor cell is intended to refer to a single cell, while reference to “circulating tumor cells” or “cluster of circulating tumor cells” is intended to refer to more than one cell.
• reference to “circulating tumor cells” is intended to include a population of circulating tumor cells including one or more circulating tumor cells.
• CTC circulating tumor cell
• CTC circulating tumor cell
• CTC circulating tumor cell
• cluster includes cancer cells, it also is intended to include non-tumor cells that are not commonly found in circulation, for example, circulating epithelial or endothelial cells.
• tumor cells and non-tumor epithelial cells are encompassed within the definition of CTCs.
• cancer includes a variety of cancer types which are well known in the art, including but not limited to, dysplasias, hyperplasias, solid tumors and hematopoietic cancers. Many types of cancers are known to metastasize and shed circulating tumor cells or be metastatic, for example, a secondary cancer resulting from a primary cancer that has metastasized. Additional cancers may include, but are not limited to, the following organs or systems: brain, cardiac, lung, gastrointestinal, genitourinary tract, liver, bone, nervous system, gynecological, hematologic, skin, breast, and adrenal glands. Additional types of cancer cells include gliomas (Schwannoma, glioblastoma, astrocytoma),
• neuroblastoma pheochromocytoma, paraganlioma, meningioma, adrenalcortical carcinoma, medulloblastoma, rhabdomyoscarcoma, kidney cancer, vascular cancer of various types, osteoblastic osteocarcinoma, prostate cancer, ovarian cancer, uterine leiomyomas, salivary gland cancer, choroid plexus carcinoma, mammary cancer, pancreatic cancer, colon cancer, and megakaryoblastic leukemia; and skin cancers including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, sarcomas such as fibrosarcoma or hemangiosarcoma, and melanoma.
• sample refers to any sample suitable for the methods provided by the present invention.
• the sample may be any sample that includes rare cells suitable for detection.
• Sources of samples include whole blood, bone marrow, pleural fluid, peritoneal fluid, central spinal fluid, urine, saliva and bronchial washes.
• the sample is a blood sample, including, for example, whole blood or any fraction or component thereof.
• a blood sample, suitable for use with the present invention may be extracted from any source known that includes blood cells or components thereof, such as veinous, arterial, peripheral, tissue, cord, and the like.
• a sample may be obtained and processed using well known and routine clinical methods (e.g., procedures for drawing and processing whole blood).
• an exemplary sample may be peripheral blood drawn from a subject with cancer.
• FIG. 3 is a functional block diagram of the overall system.
• One or more slides 20 are prepared for analysis. See the detailed description, below, for sample collection, preparation and processing description.
• Scanners 22 image the one or more slides 20.
• the scanners 22 preferably are multi-channel scanners, such as 4 color scanners.
• Data from the scanners 22 is supplied to an image store 24.
• the image store 24 may be composed of storage, preferably mass storage, such as RAID systems, as are known to those skilled in the art.
• the scanned data from the image store is provided to one or more of the technical analysis module 26, the technical analysis report module 32 and or the output 34 to a professional review, such as by a specialist, for review.
• the technical analysis module 26 serves at least to analyze the data from the image store 24 in ways described in detail, below.
• the analysis includes, but is not limited to, analyzing the cell for nuclear area or size (such as by analyzing for the intensity of blue DAPI), analyzing for the absence of CD-45 (such as by scanning for the intensity of the secondary antibody associated with CD-45 antibody, and/or analyzing for the intensity of the antibody associated with eytokeratme,
• the technical analysis report comprises an HTML file with data, including celt or object images, in the file. Automated analysis may be supplemented with analysis by a medical
• the output of the technical analysis module 26 is preferably provided to a medadata database 28.
• the metadata database includes the information generated by all of the various forms of analysis of the d ata.
• a return loop control path 30 permits the use of the scan data and analysis to then control the re -imaging of the siide(s) 20. in the event that further analysis of an object, such as a cell, is required, the system can cause the reimaging of the object.
• the degree of adhesion or adherence of the objects to the slide should be sufficient to maintain the location of the objects on the slide for at least the duration of the reimaging.
• the degree of adhesion or adherence of the objections to the slide should be sufficient to permit the subsequent identification of specific objects identified by the analysis, such as described below, for the subsequent further processing of on object, such as for genotyping or other subsequent analysis.
• the length of time for storage of the slides, and the desire that the cell location remain stationary, may range from hours to months.
• the system includes a consolidated store 40 such as for the data and reports.
• Information from the metadata database 28 is preferably provided to an data inventor;/ management system 38.
• the system comprises the management system for the overall system.
• the system 38 maintains the correlation of patient identification with the slides.
• FIG. 4 is a schematic view of one possible implementation of the scanning system.
• a stage 42 such as an x-y stage, supports one or more slides 20. For the size of well area described herein, four slides permit the scanning of approximately 10 million cells, which is a typical size sample for one patient.
• the optical path may take any form consistent with, the embodiments described herein,
• the illumination components may include light source 46, and optional excitation filter wheel 48.
• the light source is preferably a broad spectrum illuminator.
• a dichroic mirror 50 serves to pass the illumination to the slides, and to permit the return illumination io the camera 54.
• An optional emission filter wheel 52 may be placed between the mirror 50 and camera 54. The output is then stored as described above.
• FIG. 5 shows various options for the scanning of objects supported by the slide 20.
• Scanning and imaging may be in multiple dimensions, preferably in a three-dimensional frame work.
• the objects are disposed on the slide in a monolayer which is sufficiently flat to permit the scanning and imaging of the objects in an efficient manner, preferably being disposed within a single focal plane.
• the planar or fiat slide permits the imaging of the monlayer in a consistent manner as the deviation of the image plane is minimized. Imaging on a flat surface also enables easier Z-stacking of images.
• the imaging planes may be at various orientations, which may be in a non-planar relationship.
• detection of CTC candidate cells typically relies on several parameters measured from the slide imagery.
• dimensions 1-3 could be nuclear area, cytokeratine intensity and CD-45 intensity.
• the planes in FIG. 5 represent the cut-off limits for each of the measured parameters that define CTC candidates.
• light field camera systems may be utilized in which a digital camera includes light sensors which capture light rays even beyond a single focal plane, permitting the software assembly of images from various image planes.
• Lenses such as micro leases, may be used in associatio with the digital light sensors.
• FIGs. 6 and 7 define various particulars of the slide 20.
• the slide may be of any size or geometry consistent with the embodiments of the inventions described, herein.
• the slide 20 is generally rectangular in shape, with a length L s , a width W s , and a thickness t. Representative dimensions are L s of substantially 7.5 cm, W s of substantially 2.5 cm, and thickness t of 7 mm.
• the slide 20 preferably includes a top surface 72, a parallel bottom surface 74, side setbacks 66, side faces 68 and end faces 70.
• a slide identification 60 may be provided, such as via a barcode. Such slides are available from various sources, including Marienfeld Laboratory Glassware.
• a well 62 is provided to contain and maintain the materials to be imaged .
• the well 62 is rectangular and has a length L and a width W.
• Representative inner dimensions for the well 62 may be, by way of example, L of
• the periphery or perimeter of the well 62 may be defined by a border 64, whether a specific structural border or by other materials, such as by a border of hydrophobic material.
• the border may also be termed a. boundary.
• the border or boundary is adapted to receive and contain the cell suspension and al l other reagents, solutions, buffers or other liquids that are used in the process.
• the border or boundary in combination with the top side of the slide form the well. With these dimensions, the area of the well 62 is substantia] [y 1 1 .7 cm" and the perimeter of the well 62 is substantially 15.7 cm.
• the degree of setback of the well 62 from the edges of the slide 20 may be set based on other aspects of the system, such as the particulars of the scanning system.
• One embodiment provides for a single imaging well 62, providing for substantially a monolayer of objects, e.g., cells, to be imaged, having an area preferably greater than 7.5 cm , more preferably greater than 10 cm 2 , and most preferably substantially 1 1 .7 cm' ' .
• the perimeter of the well 62 is preferably substantially 12.5 cm, more preferably substantially 14.5 cm and most preferably substantially 15.7cm, correspondingly.
• the percentage of the top surface of the slide covered by the well is preferably substantially 40%, more preferably 53% and most preferably substantially 62%.
• the preferred imaging wells 62 have a total of four sides. By reducing the number of sides (as compared to the prior art, for example, the 3 well slide has .12 sides (see FIG. 1)) and their perimeter, the edge effects associated with the side wall boundaiy are minimized.
• the wells are sized so as to permit the imaging of a monolayer of preferably 1.6 to 1.9 million cells, more preferably 2.1 to 2,6 million cells, and most preferably, 2.5 to 3 million ceils, respectively.
• the slide may optionally be provided with reference marks. If the cells are sufficiently fixed in location, other structures may be used for indexing the slide. By way of example, the border may be utilized, more particularly, the 90 degree angle at the corner of the well may be used for reference.
• the apparatus and systems described herein provide for maximized and optimized cell density and minimized edge effects.
• a single cell well is utilized to hold at least 1.5 million cells, an optionally even more, such as 3 million cells.
• four side walls serve to contain that cell population.
• use of three field-slides instead of a single field slide would require use of two to three times as many slides and deal with twelve (3x4) edges on each slide instead of just four edges. Any fluid distribution suffers from edge effects no matter how hydrophobic the edges are. Cell distribution shows that the cell density at the edges goes down significantly.
• a standard sized microscope slide can be used, it is not so limited. Larger glass slides may be utilized consistent with the goals of the embodiments described herein.
• using a conventional sized slide results in process benefits by staying with a standard size, for which a large base of installed machines exist, such as automation, existing microscope systems and storage systems, to name a few.
• the system further preferably includes a single cover slide per slide.
• the system serves to optimize speed while producing sufficient quality.
• a single and very flat homogenous monolayer is preferred.
• Yet a further advantage of using a single cover slide is that a uniform surface is presented for imaging. A much more even mounting media distribution is provided using a single cover slip instead of three as would be needed in a standard three well slide.
• a sample processed as described herein includes greater than about 1, 2, 5, 7, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or even 1000 rare cells or CTCs.
• CTC analysis enables the detection of early relapse in presymptomatic patients who have completed a course of therapy. This is possible because the presence of CTCs has been associated and/or correlated with tumor progression and spread, poor response to therapy, relapse of disease, and/or decreased survival over a period of time.
• enumeration and characterization of CTCs provides methods to stratify patients for baseline characteristics that predict initial risk and subsequent risk based upon response to therapy.
• subject refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
• rodents including mice, rats, hamsters and guinea pigs
• cats dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc.
• primates including monkeys, chimpanzees, orangutans and gorillas
• the invention provides a method for diagnosing or prognosing cancer in a subject.
• the method includes detecting CTCs as described herein. CTCs may then be analyzed to diagnose or prognose cancer in the subject.
• the methods of the present invention may be used, for example, to evaluate cancer patients and those at risk for cancer.
• either the presence or the absence of one or more indicators of cancer, such as, a cancer cell, or of any other disorder may be used to generate a diagnosis or prognosis.
• a blood sample is drawn from the patient and processed to detect CTCs as described herein.
• the number of CTCs in the blood sample is determined and the CTCs are characterized by analysis of the detectable markers and other data gathered from imaging the cells. For example, analysis may be performed to determine the number and characterization of CTCs in the sample, and from this measurement, the number of CTCs present in the initial blood sample may be
• analysis of a subject's CTC number and characterization may be made over a particular time course in various intervals to assess a subject's progression and pathology. For example, analysis may be performed at regular intervals such as one day, two days, three days, one week, two weeks, one month, two months, three months, six months, or one year, in order to track level and characterization of circulating epithelial cells as a function of time. In the case of existing cancer patients, this provides a useful indication of the progression of the disease and assists medical practitioners in making appropriate therapeutic choices based on the increase, decrease, or lack of change in circulating epithelial cells, such as the presence of CTCs in the patient's bloodstream.
• Any decrease, be it 2-fold, 5- fold, 10-fold or higher, in the number of CTCs over time shows disease stabilization and a patient's response to therapy, and is an indicator to not change therapy.
• a sudden increase in the number of CTCs detected may provide an early warning that the patient has developed a tumor thus providing an early diagnosis.
• the detection of revealed CTCs increases the staging of the cancer.
• additional analysis may also be performed to characterize CTCs, to provide additional clinical assessment.
• gene expression analysis and PCR techniques may be employed, such as gene chip analysis and multiplexing with primers specific for particular cancer markers to obtain information such as the type of tumor, from which the CTCs originated, metastatic state, and degree of malignancy.
• cell size, DNA or R A analysis, proteome analysis, or metabolome analysis may be performed as a means of assessing additional information regarding characterization of the patient's cancer.
• analysis includes antibodies directed to or PCR multiplexing using primers specific for one or more of the following markers: EGFR, HER2, ERCC1, CXCR4, EpCAM, E-Cadherin, Mucin- 1, Cytokeratin, PSA, PSMA, RRMl, Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1, cMET, EML4, or Leukocyte Associated Receptor (LAR).
• markers include antibodies directed to or PCR multiplexing using primers specific for one or more of the following markers: EGFR, HER2, ERCC1, CXCR4, EpCAM, E-Cadherin, Mucin- 1, Cytokeratin, PSA, PSMA, RRMl, Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1, cMET, EML4, or Leukocyte Associated Receptor (LAR).
• the additional analysis may provide data sufficient to make determinations of responsiveness of a subject to a particular therapeutic regime, or for determining the effectiveness of a candidate agent in the treatment of cancer.
• the present invention provides a method of determining responsiveness of a subject to a particular therapeutic regime or determining the effectiveness of a candidate agent in the treatment of cancer by detecting CTCs of the subject as described herein and analyzing the detected CTCs. For example, once a drug treatment is administered to a patient, it is possible to determine the efficacy of the drug treatment using the methods of the invention.
• a sample taken from the patient before the drug treatment, as well as one or more cellular samples taken from the patient concurrently with or subsequent to the drug treatment, may be processed using the methods of the invention. By comparing the results of the analysis of each processed sample, one may determine the efficacy of the drug treatment or the responsiveness of the patient to the agent. In this manner, early identification may be made of failed compounds or early validation may be made of promising compounds.
• Four important indicators that provide insight to the clinical activity of candidate compounds include HER2, EGFR, CXCR4, and EphB4 RTK.
• HER2 provides an indicator of malignancy of a cell by determining mRNA stability and subcellular localization of HER2 transcripts.
• the resistance of EGFR to acquire mutations, and/or the mutations acquired provides important indicators of the activity of a candidate compound in addition to possible alternative compounds that may be used in combination with the candidate compound.
• An assessment of the level of DNA repair interference induced with platinum provides insight as to the status of the CXCR4 marker and metastatic condition. Additionally, assessment of the status of EphB4 receptor tyrosine kinase provides insight as to the metastatic potential of the cell. Accordingly, using the methods of the present invention, patients taking such candidate drugs may be monitored by taking frequent samples of blood and determining the number of circulating epithelial cells, for example CTCs, in each sample as a function of time.
• Her2, EGFR, CXCR4, and EphB4 RTK indicators provides information as to pathology of the cancer and efficacy of the candidate drug.
• ERRCl Cytokeratin, PSA, PSMA, RRMl , Androgen Receptor, Estrogen Receptor, Progesterone Receptor, IGF1 , cMET, EML4 and others provide insight into the clinical activity of candidate compounds.
• the analysis of these indicators of clinical activity may be through analysis of detectable markers as discussed herein (e.g., immunohistochemistry and fluorescent in situ hybridization (FISH)) or further analysis via techniques such as sequencing, genotyping, gene expression or other molecular analytical technique.
• FISH fluorescent in situ hybridization
• Analysis of CTCs provide a method of determining candidate subjects for a particular clinical trial.
• the detected CTCs of a candidate may be analyzed to determine whether specific markers exist in order to determine whether the particular therapeutic regime of the clinical trial may be potentially successful.
• the invention provides a method for determining a candidate subject for a clinical trial. The method includes detecting CTCs of the subject as described herein. The CTCs may then be analyzed to determine whether the candidate subject is suitable for the particular clinical trial.
• the system preferably defines one or more measures for the cells to be utilized in the analysis.
• Various approaches define an intact CTC based on the investigation of large numbers of candidate events in patients with epithelial cancers, with direct comparison to cell from the solid forms of the same tumor in the same patient [9-12]. Based on these definitions, and utilizing the HD-CTC assay to refine criteria, a definition of an HD-CTC was established. This definition has been developed to ensure that an HD-CTC is a cell that has the highest potential of being an intact cell originating from a solid deposit of carcinoma in the patient's body.
• CTCs may be morphologically characterized and credentialed in relation to their primary tumors, in case studies of breast, colorectal, and lung cancer patients. Through morphologic examination of CTCs in a patient with a well differentiated lung
• circulating cells were identified with morphologic features consistent with this type of tumor, including, for example, cells with relatively low nuclear-to-cytoplasmic ratios.
• the morphology of the tumor cells identified in circulation mimicked the morphology found in that patient's fine needle aspirate biopsy of the primary tumor [9].
• HD-CTCs are classified as cells that are a) cytokeratin positive; b) CD45 negative, with an intact non- apoptotic appearing nucleus by DAPI imaging.
• Positivity for CK is defined as the fluorescent signal being significantly above the signal of surrounding ceils. Negativity is defined as being the same level or below the signal of the surrounding cells.
• Negativity for CD45 is defined as having intensity beiow visual detection under the boundar condition that 99% of ail cells are detectable globally, A gallery of representative HD-CTCs found in cancer patients is not shown, however, HD-CTCs are cytokeratin positive, CD45 negative, contains a DAPI nucleus, and are morphologically distinct from surrounding white blood cells.
• HD-CTCs must be morphologically distinct from normal WBCs, and must have a morphology that is compatible with a malignant cel l by criteria used in standard diagnostic cytopathology, predominantl embodied as enlarged size, but also encompassing c tomoiphologic features such as architectural organization of nucleus and cytoplasm, cytoplasmic shape, and nuclear shape.
• a lower nuclear size cut-off of 1.3 times the mean WBC nuclear size may be set.
• Table 1 Percentage of patients with HD-CTCs per 1 mL of blood obtained from metastatic prostate, breast, and pancreatic cancer patients as well as normal controls.
• HD-CTCs were enumerated in. a cohort of 30 metastatic breast cancer patients, 20 metastatic prostate cancer patients, 18 metastatic pancreatic cancer patients, and 15 normal controls. The incidence of D-CTCs in the three types of cancers investigated is displayed in
• FIG. 8 shows mean observed SKBR3s plotted against expected SKBR3s.
• Four aliquots of normal control blood was spiked with varying numbers of SKBR2 cells to produce 4 slides with approximately 10, 30, 100, and 300 cancer cells per slide.
• the mean of each quadruplicate is displayed as well as error bars noting standard deviation.
• SKBR3s were spiked into normal control blood in quadruplicates and processed according to the HD-CTC assay. As displayed in FIG. 8, mean observed SKBRSs is plotted against expected SKBR3s and displays a correlation coefficient (R") of 0.9997.
• FIG. 9 is a comparison of CTC counts between two separate processors on 9 different cancer patient samples. CTC/mL counts ranged from 0 to 203.
• Explicit re-review of these cells revealed a similar pattern, in that about one third strongly met all criteria, while the remaining two thirds of the cells fulfilled criteria, but were near the lower limit for inclusion by one or more criteria.
• Examples of the latter type of event include cells that measure 30% larger than surrounding WBCs but do not appear significantly larger by morphologic evaluation, and cells that are slightly out of focus and might have apoptotic nuclear changes that are not detectable by eye, and finally, occasional cells that have objective cytokeratin intensity measurements above the cutoff but subjectively don't appear significantly brighter than surrounding WBCs by single channel fluorescent review.
• the CeliSearch® assay detected 2 or more CTCs per 7.5mL of blood in 5/15 patients tested. In contrast, the HD-CTC assay detected significantly higher numbers of CTCs in significantly more patients (HD-CTCs were identified in 14/15 patients tested). [0085] Table 2: Comparison of HD-CTC Assay to CellSearch® from 5 metastatic breast cancer patients and 10 metastatic prostate cancer patients. CellSearch® values are extrapolated to number of CTCs per mL of blood.
• HD-CTCs had various shapes, sizes, and cytokeratin intensities, in some cases, distinctive cytologic features such as large size or polygonal cytoplasmic shape were quite distinctive and monotonous within the patient ' s sample. In other eases, there was cytomorphologic variability between HD-CTCs within a single sample. Ceil size also varied; many patient samples had HD-CTCs with nuclei uniformly three or four tim.es the size of neighboring WBC nuclei, while other patients had cells with nuclei uniformly only 1.3 times the size of neighboring WBC nuclei. Some patients had a range of sizes.
• a lower limit criterion was seSected for HD-CTC nuclear size of 1.3 times the average WBC nucleus, based on evaluation of the largest nuclear size of cells identified as WBCs showing false nonspecific staining wit cytokeratin (i.e. CD45 positive and cytokeratin positive).
• HD-CTC doublets and clusters were identified in the majority of the cancer patients in this cohort (88%), ranging from clusters of 2 HD-CTCs to greater than 30 HD-CTCs (data not shown). [0089] Clusters were found in most patients with cancer. Clusters ranged from 2 to over 30 HD-CTCs. Each HD-CTC was determined to be cytokeratin positive, CD45 negative, contain a DAPI nucleus, and was morphologically distinct from surrounding nucleated cells.
• cytokeratin intensity was too dim; the nuclear size was too small; cytokeratin was observed to be insufficiently circumferential (surrounds less than 2/3 of nucleus); observed cytokeratin was too dim, even though cluster appeared to be of multiple large cells; nucleus showed apoptotic disintegration changes; nucleus was too small and cytoplasm was insufficiently circumferential; appeared to be a cell in late apoptosis; nucleus was too small (same size as surrounding WBC nuclei); cytokeratin was present, but not circumferential; and cytoplasm was insufficiently circumferential, and the nucleus was too small.
• Various types of suspected CTCs were also found in a single prostate cancer patient. For example, some were negative for Cytokeratin and CD45, but had a nucleus that was large and looked like other HD-CTCs found in this patient. Typical HD-CTC were also observed in which the cells were cytokeratin positive, CD45 negative, with a DAPI nucleus. Clusters of HD-CTCs of multiple cells, e.g., 4 cells were also observed. [0095] In light of the extensive current debate about the possible existence of carcinoma cells undergoing epithelial-to-mesenchymal transition, the appearance and protein expression pattern of these cells identifies them as possible candidates for such a cell type.
• these cells could be older tumor cells that have been stripped of most of their cytoplasm.
• CTCs had various shapes, sizes, and cytokeratin intensities. In some cases, distinctive cytologic features such as large size or polygonal cytoplasmic shape were quite distinctive and monotonous within the patient's sample. In other cases, there was
• cytomorphologic variability between HD-CTCs within a single sample cytomorphologic variability between HD-CTCs within a single sample.
• Cell size also varied inconsistently; many patient samples had HD-CTCs with nuclei uniformly three or four times the size of neighboring WBC nuclei, other patients had cells with nuclei only a third again as large as neighboring WBC nuclei, and other patients had high intra-patient size variability.
• the cohort of patients demonstrated the common occurrence of clusters of HD-CTCs in most patients. 88% of the metastatic cancer patients evaluated in this cohort study showed clusters ranging in size from 2-30 HD-CTCs. Multiple questions arise around the presence of such clusters, including the rheology of transit through the circulatory system of such a large aggregate, as well as biological questions about whether such clusters represent 'tumorlets' that are transporting their own microenvironmental stroma with them as they travel and thus may be the most, or only, truly metastable circulating tumor cells.
• CTC-like cells were independently tracked, including cells that had nuclei displaying apoptosis, cells lacking circumferential cytokeratin, cells that were the same size or smaller than surrounding WBC, and CD45-negative cells that were cytokeratin dim or negative (data not shown). Although many of these events may in fact represent circulating malignant epithelial cells in various stages of biologic anoikis or mechanical disruption secondary to even the minimal processing utilized in the platform, others likely represent false positives of various types. An initial goal is to identify a population of cells with a very high likelihood of including all potentially metastasizing epithelial cells that are suitable for downstream analysis by secondary methodologies.
• Fragmented, disrupted, pyknotic or otherwise damaged carcinoma cells are not considered reliable for secondary analysis in standard diagnostic pathology, and thus they were excluded for purposes of 'counting viable circulating tumor cells' in this fluid phase biopsy platform as well.
• the systems, apparatus and methods of these embodiments locate, enumerate and track them, as it is recognized that their presence likely correlates overall with the tumor biology in the patient, either by reflecting overall tumor burden or by reflecting some as yet ill-understood complex equation involving tumor burden and tumor vascularity and efficiency of intravascular immune surveillance.
• One of the interesting non-HD-CTC categories consists of cells with nuclei that are morphologically distinct from surrounding WBC, generally by size criteria, and were CD45 negative, but are also cytokeratin dim or negative.
• the HD-CTC assay is that parallel aliquots of cells are frozen, allowing for retrospective marker selection in specific high-yield patient samples, ongoing studies to further characterize such cells is in progress. Possibilities include epithelial cells with denatured or stripped cytoplasm, cells aberrantly expressing or aberrantly lacking proteins typical for their biologic origin, or possibly cells undergoing a metaplastic process such as epithelial to mesenchymal transition.
• the assay and imaging platforms are currently limited to analysis of fixed cells; however efforts are underway to establish the potential utility of this approach for live cell enumeration and imaging.
• FIG. 10 shows comparative test data of the systems, apparatus and methods described here, versus the CellSearch® product.
• the left most column identifies five breast cancer tumors and for 10 prostate cancer tumors.
• the second column states the mL/test.
• the third column shows the observed CTCs using the systems, apparatus and methods of the described embodiment.
• the fourth column provides the calculated CTCs/mL.
• the two rightmost columns provide the comparative data for the CellSearch® product, reported as per 7.5 ml (as compared to per mL in the fourth column.)
• FIG. 11 shows test results graphing the quantity of CTCs for various samples, for prostate, pancreatic, breast tumors, and a comparison to healthy population. From left to right are data for prostate cancer, pancreatic cancer, breast cancer and for a presumed healthy population. These results provide the number of CTCs/ml by sample observed CTCs using the systems, apparatus and methods of the embodiments described herein.
• FIG. 12 shows the quantity of CTCs for various patient samples relative to breast cancer.
• the left-most graph shows HER2-, not on Herceptin and HER2+, Herceptin.
• the center graph shows a comparison of not on Herceptin (left) with on Herceptin (right).
• the right-most chart shows HER2 negative (left) versus HER2 positive (right)
• FIG. 13 shows the normalized nuclear area versus nuclear area for white blood cells (WBCs) and CTCs, including a blow-up of the base-line region.
• the left axis in the underlying graph is from 0 to 700,000.
• the blow-up is from 0 to 400.
• FIGs. 16 and 17 show that a single parameter such as nuclear area may not reduce the number of candidates on a slide to a tractable amount. While is may appear that the use of a lower limit on nuclear size would remove most of the noise (WBCs), the blow-up shows that the number of non-CTC candidates is still large compared to the actual HD-CTCs.
• the use of more parameters, such as CK intensity and CD-45 intensity serves to effectively filter out the non-HD CTC events.
• the HD-CTC assay finds significant number of CTCs in most patients with metastatic cancer, (ii) has improved sensitivity over the Celisearch® system, (iii) provides HD-CTCs in an ideal format for downstream characterization, (iii), enables the prospective collection of samples that can be stored frozen for long periods of time and then retrospectively analyzed as new assays or markers become available.
• Samples were col lected from metastatic cancer patients in anti -coagulated blood tubes at Scripps Clinic. University of California, San Diego, Billings Clinic, and University of California, San Francisco under Institutional Review Boards (IR B) approved protocols. Samples from non-local sites (UCSF, Billings Clinic) were shipped overnight so that the sample was received and processed within 24 hours. Samples from local sites (Scripps Clinic and UCSD) were held at room temperature for 16-24 hours to mimic samples coming from non-local sites. Blood specimens were also drawn from normal controls from the The Scripps Research Institute ("TS J") Normal Blood Donor Service.
• TS J The Scripps Research Institute
• All likely candidate CTCs are presented to a hematopathologist for analysis and interpretation through a web based report where the pathologist is able to include or exclude each candidate cell as an HD-CTC.
• Cells are classified as HD-CTCs if they are cytokeratin positive, CD45 negative, contained an intact DAPI nucleus without identifiable apoptotic changes (blebbing, degenerated appearance) or a disrupted appearance, and are
• cytoplasm morphologically distinct from surrounding white blood cells (usually a shape based feature, although occasionally purely size based.) They must have cytoplasm that is clearly circumferential and within which the entire nucleus is contained. The cytoplasm may show apoptotic changes such as blebbing and irregular density or mild disruption at the peripheral cytoplasmic boundary, but must not be so disrupted that its association with the nucleus is in question.
• the images are presented as a digital image, with individual fluorescent channel viewing capability as well as a composite image. Each cell image is annotated with ancillary statistical data regarding relative nuclear size, fluorescent intensities, and comparative fluorescent intensities.
• Each HD-CTC candidate is presented in a field of view with sufficient surrounding WBCS to allow for contextual comparison between cytomorpho logic features of the cell in question versus the background white blood cells.
• the HD-CTC assay was specifically developed with the clinical environment in mind as well as the need for early technology innovation and future automation. All laboratory processes follow strict standard operating procedures that have been optimized, tested and validated. Data collection and candidate identification has been automated using specific interfaces that both enable the pathologist's decision making and subsequent tracking of these decisions. Specifications for both complete automation and adaption to routine settings will arise from this early research framework.

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