HK1202923B

HK1202923B - Methods for detecting 5t4-positive circulating tumor cells and methods of diagnosis of 5t4-positive cancer in a mammalian subject

Info

Publication number: HK1202923B
Application number: HK15103408.3A
Authority: HK
Inventors: H-P‧格贝尔; D‧马里努奇; S‧皮里谢泼德; E‧塔克
Original assignee: 辉瑞公司; 艾匹克科学公司
Priority date: 2012-01-24
Filing date: 2013-01-22
Publication date: 2018-05-11

Description

Methods of detecting 5T4 positive circulating tumor cells in a mammalian subject and methods of diagnosing 5T4 positive cancers

Technical Field

The present invention relates generally to methods of detecting 5T4 positive circulating tumor cells in a mammalian subject, and methods of diagnosing 5T4 positive cancer in a mammalian subject.

Background

The human 5T4 antigen is expressed in many cancer types and is essentially absent from normal tissues. Recently, high affinity monoclonal antibodies that specifically bind to the 5T4 antigen have been developed, and cytotoxic drugs have been conjugated to the 5T4 antibody to form antibody drug conjugates for the treatment of 5T 4-positive cancers (U.S. patents 8,044,178 and 8,309,094). Subsequent assessment of expression of 5T4 may be a useful method to identify patients with 5T4 positive cancer. One approach is to detect the 5T4 antigen on Circulating Tumor Cells (CTCs) of cancer patients.

Circulating tumor cells have been observed at ultra-low concentrations in peripheral blood of patients with epithelial-derived cancers (Kraeft et al, Clin Cancer Res10:3020-3028, 2004). The number of these cells when sampled was shown to correlate with the outcome in multiple groups of metastatic breast cancer patients with progressive disease (Cristofanlli et al, N Engl JMed351: 781-. For this reason, the characterization of these cells is of great biomedical interest in order to study how these cells automatically reach remote sites via the bloodstream and form metastatic disease. Thus, identification of CTCs associated with 5T 4-positive cancers may provide a valuable diagnostic tool for patient identification.

CTCs are currently detected and analyzed mainly by immunocytochemical markers such as EpCam and nuclear staining with DAPI (4', 6-diamidino-2-phenylindole), a fluorescent dye that binds strongly to the a-T rich region of DNA. Although these methods have been successfully used to calculate and differentiate CTCs, they differ from standard cytopathological methods in that they ignore the correlation with standard morphological staining on which diagnostic pathology relies. This poses difficulties in comparing CTCs to tumor cells at other sites obtained by conventional diagnostic methods. Although the ability to detect CTCs may aid in the diagnosis and personalized treatment of cancer as well as improving the efficiency of treatment, by including standard cytopathological approaches, it is possible to improve understanding of CTC biology. There is a need in the art to utilize detailed high resolution CTC imaging and conventional diagnostic pathology staining methods and bright field microscopy to enable the following clinical applications: standard cytopathological diagnosis of circulating 5T 4-positive cancer cells and facilitates clinical adoption of diagnosis using 5T 4-positive CTCs.

Summary of The Invention

In one embodiment, the invention provides a method of detecting 5T4 positive circulating tumor cells in a mammalian subject suspected of having a 5T 4-positive cancer, comprising: testing a blood sample of the individual, wherein the blood sample comprises a population of cells; placing the blood sample on a substrate; detecting the presence or absence of a first marker that selectively binds to nucleated cells in the blood sample; detecting the presence or absence of a second marker in said blood sample that binds to circulating tumor cells; detecting the presence or absence of a third marker in the blood sample that binds to a cell population or subset of cell populations determined to not be tumor cells; detecting the presence or absence of a fourth marker in said blood sample that selectively binds to circulating tumor cells, said fourth marker being human 5T4 antigen; and analyzing the cell populations detected by the first, second, third, and fourth markers to identify and characterize circulating tumor cells.

In another embodiment, a method of detecting the presence of 5T4 positive circulating tumor cells in a mammalian subject suspected of having a 5T 4-positive cancer indicates that the mammalian subject has an early stage 5T 4-positive cancer, is in a disease-free state, or is in a non-measurable disease state.

In another embodiment, the presence or absence of circulating tumor cells in the blood sample is indicative of 5T 4-positive cancer treatment or treatment management during cancer recovery.

In another embodiment, the cell population is a mixed cell population and the substrate is a planar substrate, microfluidic device, or cartridge (cartrige) containing an enriched cell population.

In another embodiment, the test sample is placed on a substrate to form a biological monolayer.

In another embodiment, the population of cells is analyzed by nuclear details, nuclear contour, presence or absence of nucleoli, cytoplasmic character or amount of cytoplasm, wherein the analysis uses DAPI.

In another embodiment, the cell population is analyzed by measuring intact cells with a high nuclear to cytoplasmic ratio, intact cells with a low nuclear to cytoplasmic ratio, early apoptotic cells, or late apoptotic cells, and identifying circulating tumor cells.

In another embodiment, the first label, the second label, the third label, and the fourth label are fluorescent labels.

In another embodiment, the first marker is a cellular dye to identify circulating tumor cells by morphology, size, or nuclear to cytoplasmic ratio.

In another embodiment, the cellular dye is DAPI.

In another embodiment, the cellular dye is a Giemsa rapain dye (Wright-Giemsa stain).

In another embodiment, the second marker or the third marker is a cell-specific marker.

In another embodiment, the cell-specific marker is cytokeratin, CD45, M30, chemokine receptor, CXCR1, CXCR4, CD44, CD24, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, or HuR.

In another embodiment, detecting the presence of the first label, the presence of the second label, the presence of the third label, or the presence of the fourth label further comprises analyzing the population of cells by their attachment to a substrate, scanning the population of cells on the substrate, and imaging the cells by digital microscopy using repositioning.

In another embodiment, detection of 5T 4-positive circulating tumor cells in the blood sample indicates the presence of a 5T 4-positive cancer, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, renal cancer, liver cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, and testicular cancer. Preferably, the cancer is selected from colorectal cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer.

In another embodiment, the invention provides a method of diagnosing a 5T 4-positive cancer in a mammalian subject suspected of having a 5T 4-positive cancer, comprising: testing a blood sample of the individual, wherein the blood sample comprises a population of cells; placing the blood sample on a substrate; detecting the presence or absence of a first marker that selectively binds to nucleated cells in the blood sample; detecting the presence or absence of a second marker in said blood sample that binds to circulating tumor cells; detecting the presence or absence of a third marker in the blood sample that binds to a cell population or subset of cell populations determined to not be tumor cells; detecting the presence or absence of a fourth marker in the blood sample that selectively binds to circulating tumor cells, wherein the fourth marker is human 5T4 antigen; and analyzing and quantifying the cell populations detected by the first, second, third and fourth markers to identify and characterize circulating tumor cells.

In another embodiment, the invention provides a method wherein the quantification of human 5T4 antigen on circulating tumor cells is used to generate an H-score, wherein the H-score is used to select a population of 5T4 positive cancer patients, and wherein the circulating tumor cells are characterized using an optimized 5T44 color analysis.

In another embodiment, the present invention provides a method of screening for an antibody drug conjugate for use in treating a mammalian subject suspected of having cancer for a 5T 4-positive cancer, comprising: administering to an individual suspected of having cancer a therapeutically effective amount of an antibody drug conjugate; testing a blood sample of the subject before and after treatment with the drug candidate, wherein the blood sample comprises a population of cells suspected of containing 5T4 positive circulating tumor cells; placing the blood sample on a substrate; detecting the presence or absence of a first marker that selectively binds to nucleated cells in the blood sample; detecting the presence or absence of a second marker in said blood sample that binds to circulating tumor cells; detecting the presence or absence of a third marker in the blood sample that binds to a cell population or subset of cell populations determined to not be tumor cells; detecting the presence or absence of a fourth marker in the blood sample that selectively binds to circulating tumor cells, wherein the fourth marker is human 5T4 antigen; and analyzing the cell populations detected by the first, second, third, and fourth markers to identify circulating tumor cells in the blood sample before treatment with the antibody drug conjugate as compared to after treatment with the antibody drug conjugate, wherein a change in the ratio of 5T4 positive circulating tumor cells to 5T4 negative circulating tumor cells in the blood sample after treatment as compared to the ratio of 5T4 positive circulating tumor cells to 5T4 negative circulating tumor cells in the blood sample before treatment may indicate the efficacy of the antibody drug conjugate in reducing 5T4 positive circulating tumor cells, wherein the antibody drug conjugate compound is anti-5T 4-a 1-mcMMAF.

In another embodiment, the invention provides a method of detecting 5T4 positive circulating tumor cells in a mammalian subject suspected of having a 5T 4-positive cancer comprising: testing a blood sample of the individual, wherein the blood sample comprises a population of cells; placing the blood sample on a substrate; detecting the presence or absence of a first marker that selectively binds to nucleated cells in the blood sample, wherein the first marker is DAPI; detecting the presence or absence of a second marker in said blood sample that binds to circulating tumor cells, wherein said second marker is cytokeratin; detecting the presence or absence of a third marker in said blood sample that binds to a cell population or subset of a cell population determined to not be a tumor cell, said third marker being CD 45; detecting the presence or absence of a fourth marker in said blood sample that selectively binds to circulating tumor cells, said fourth marker being human 5T4 antigen; and analyzing the cell populations detected by the first, second, third, and fourth markers to identify and characterize circulating tumor cells.

Drawings

FIG. 1a compares the expression ranges of 5T4 used to calculate the H score.

FIG. 1b provides a corrected cell line depicting the thresholds established for low, medium and high expression of 5T4 in non-small cell lung cancer (NSCLC).

Figure 2a shows a scatter plot of 5T4 expression of single CTCs and CTC clusters from a NSCLC patient sample analyzed with an optimized 5T 44-color diagnostic assay.

Figure 2b shows data from 17 NSCLC patient samples analyzed using an optimized 5T 44-color diagnostic analysis and H-scores calculated using the corrected cell line.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

5T4 refers to 5T4 carcinoembryonic antigen, a 72 kDa highly glycosylated transmembrane glycoprotein comprising a 42 kDa non-glycosylated core (see U.S. Pat. No.5,869,053). Human 5T4 is expressed in many cancer types including, but not limited to, bladder, breast, cervical, colon, endometrial, kidney, lung, esophageal, ovarian, prostate, pancreatic, skin, stomach, and testicular cancer. High tumorigenic cells, also called cancer stem cells or tumor initiating cells, were shown to have high levels of 5T4 expression (WO 2010/111659). anti-5T 4 antibodies include antibodies that specifically bind to human 5T4 antigen (see US Pat. No.8,044,178).

"biological monolayer" refers to a blood sample that can exist in a variety of cell isolated or purified states. For example, the biological monolayer may be partially purified and contain mononuclear cells and other cells after lysis of the red blood cells.

"sorting a cell population prior to placing a sample on a substrate" refers to removing a subset of the cell population from a sample, such as a blood sample. Sorting may be performed using selective cell lysis and centrifugation of a sub-fraction of the cells. Sorting can also be performed using fluorescent cell labeling and fluorescence activated cell sorting. Cell sorting for cell markers can be performed as a positive selection for circulating tumor cells or as a negative selection for removal of non-tumor cells.

The "matrix" contains a test sample, such as a blood sample containing cells, which is placed for detection and analysis. In one aspect, the substrate may be planar. In another aspect, the substrate may have some curvature.

"subject," "mammalian subject," or "patient" refers to any mammalian patient or subject to which the methods of the invention are applicable. "Mammal" or "Mammal" refers to human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, and mice, and other animals. In exemplary embodiments of the invention, to identify an individual patient for treatment by the methods of the invention, an acceptable screening method is employed to determine risk factors associated with a targeted or suspected disease or disorder (e.g., 5T 4-positive cancer), or to determine the status of an individual's existing disease or disorder. These screening methods include, for example, routine disease testing to determine risk factors that may be associated with the targeted or suspected disease or disorder. These and other conventional methods allow a clinician to select patients in need of treatment using the methods and prescriptions of the present invention.

"blood sample", "blood specimen", "test sample" and "blood sample" are used interchangeably and are defined as a quantity of blood that is typically removed or collected from an individual by: venipuncture or venipuncture percutaneously using a sharp rigid probe or cannula with a flexible plastic catheter or a steel needle attached to a syringe or catheter for medical testing including diagnostic analysis.

"cancer," "malignancy," "solid tumor," or "hyperproliferative disorder" are used synonymously and refer to any of a number of diseases characterized by uncontrolled, abnormal proliferation of 5T 4-positive cells, the ability of affected 5T 4-positive cells to spread locally or to spread through the bloodstream, lymphatic system to other parts of the body (i.e., metastases), and any of a number of characteristic structural and/or molecular features.

The "first label", "second label", "third label" and "fourth label" identify circulating tumor cells by a cell dye or by a cell-specific label. The first marker is a cellular dye including, but not limited to, DAPI, giemsa rapi dye, or other cellular dyes known in the art. See, e.g., B.F. Atkinson, Atlas of diagnostic cytopathic.2^ndEdition, w.b. saunders Company, ed.,2003, which is incorporated herein by reference in its entirety. The second and third markers are cell-specific markers including, but not limited to, the following for the following molecules: cytokeratin, CD45, M30, chemokine receptor, CXCR1, CXCR4, CD44, CD24, vascular endothelial growth factor isoforms (VEGFR-1, VEGFR-2, VEGFR-3), Epithelial Growth Factor Receptor (EGFR), or mRNA stability factor HuR. The fourth label refers to the 5T4 antigen.

These markers identify various cell types, including cells of hematopoietic origin, cytokeratins on epithelial cells, breast cancer cells, prostate cancer cells, CD44, cell surface receptors that recognize hyaluronic acid, chemokine receptors such as CXCR1 or CXCR 4.

"sorting" in the context of cells as used herein refers to physical sorting of cells that can be accomplished using, for example, a fluorescence activated cell sorter, as well as cell analysis based on expression of cell surface markers, such as FACS analysis without sorting.

Analysis of cell populations by nuclear details, nuclear contour, presence or absence of nucleoli, quality of cytoplasm or amount of cytoplasm "and analysis of cell populations and identification of circulating tumor cells by measuring intact cells with high nuclear to cytoplasmic ratio, intact cells with low nuclear to cytoplasmic ratio, early apoptotic cells or late apoptotic cells" can be performed using the techniques and assays described in the article by b.f. atkinson.

"management of cancer treatment or cancer recovery" refers to in vivo or in vitro diagnostic tests to determine the stage of cancer progression or the efficacy of a particular cancer therapy.

"Circulating Tumor Cells (CTCs)" refers to intact tumor cells or tumor cell clusters that are pan cytokeratin (pan cytokeratin) positive and CD45 negative. CTCs also include cells that are 5T4 positive but CD45 negative, broad spectrum cytokeratins and cells that are 5T4 positive and CD45 negative, as well as cells that are morphologically consistent with malignant cells. Methods for classification and detection of CTCs have been previously reported (WO2011/028905, WO2011/050103, and US2009/0317836, incorporated herein by reference).

An "H-score" is a weighted score that multiplies the percentage of CTCs within each category (low, medium, high) by their respective category value, and then sums to give a score of 0-300.

Several methods are known in the art for detecting circulating tumor cells. The concentration of malignant epithelial cells in the blood sample was low, amounting to 10⁶-10⁷About one malignant epithelial cell among the nucleated cells, which makes them difficult to detect. Several methods have been attempted to detect and calculate CTCs including PCR, flow cytometry, image-based immunological methods, immunomagnetic techniques, microfluidic techniques, andmicrochip technology.

For example, AdnaTest BreastThe system utilizes reverse transcriptase-polymerase chain reaction (RT-PCR) to detect circulating tumor cells (Adnagen AG, Langenhagen, Germany; OncoVista, Inc., San Antonio, TX). The test features a CTC enrichment method that utilizes a mixture of proprietary immunomagnetic beads coated with one of three antibodies to epithelial surface antigens. The number of CTCs was then determined indirectly by a semi-quantitative RT-PCR method.

CellSearch System^TM(Veridex LLC, Warren, NJ) was developed for the detection of CTCs in whole blood. The CellSearch system involves a technique of mixing a blood sample with iron particles coated with proteins that attach to epithelial cells. The epithelial cells are then distinguished from the leukocytes by antibodies that have been labeled with fluorescent dyes, so that the cancer cells can be easily distinguished and counted.

OncoQuick^TM(Greiner Bio-One-, Inc. Longwood, FL) is another test system developed to detect circulating tumor cells. The system is an enhanced density gradient system that combines density gradient centrifugation and immune-based techniques.

In U.S. patent application No. 2010/0233693a (incorporated herein by reference), a method of calculating the number of CTCs in a sample from a patient is described that includes flowing the sample through a microfluidic device that is selectively enriched for one or more circulating tumor cells. The microfluidic device may enrich one or more CTCs based on size, affinity, deformability, or shape.

A method for separating and analyzing CTCs using a microchannel device is described in U.S. patent application No. 2010/0255479. The method provides for capturing biological targets from solution by pre-labeling or pre-mixing a sample containing CTCs with a binding partner that specifically binds to the cells, thereby enhancing capture of CTCs in a microchannel device.

Each of the above methods for detecting circulating tumor cells requires a cell enrichment step. A distinguishing feature of the present invention is the absence of an enrichment assay that demonstrates the ability to identify large numbers of CTCs in a majority of patients with 5T 4-positive cancer.

One aspect of the invention generally relates to a method of detecting 5T4 positive Circulating Tumor Cells (CTCs) in a mammalian subject or a method of diagnosing early stage 5T4 positive cancer in a mammalian subject. The present invention also relates to methods of screening for drug candidate compounds for treating 5T4 positive cancer in a mammalian subject.

A method for detecting 5T4 positive CTCs in a mammalian individual is provided, comprising obtaining a blood sample comprising a mixed population of cells suspected of containing CTCs from a mammalian individual suspected of having cancer, placing the blood cells and CTCs on a substrate to form a biological monolayer, detecting a first marker in the biological monolayer that selectively binds to nucleated cells, detecting a second marker in the biological monolayer that binds to CTCs, detecting a third marker in the biological monolayer that binds to the mixed population or a subset of the mixed population, detecting a fourth marker in the biological monolayer that selectively binds to 5T4 positive cells, analyzing the cell populations detected by the first, second, third, and fourth markers to identify CTCs; the presence of CTCs in the blood sample indicates the presence of a 5T 4-positive cancer or an early 5T 4-positive cancer in the mammalian individual. The presence or absence of CTCs in the blood sample can indicate the presence of a disease-free state or a non-measurable disease state in the mammalian subject.

The methods provide cell attachment protocols to identify cells of epithelial origin in blood samples, as well as methods of detecting 5T4 positive CTCs in blood of cancer patients. In this protocol, live White Blood Cells (WBCs) such as leukocytes (leukocytes) and other cells in blood are separated on a slide, for example, as a biological monolayer. Leukocytes include, but are not limited to: t-lymphocytes, monocytes, eosinophils, and neutrophils involved in phagocytosis; and basophils involved in inflammatory responses.

The method also provides fluorescently labeled attached WBCs and CTCs on specially coated adherent slides. Cells are fluorescently labeled with a first label that selectively binds nucleated cells, a second label that binds circulating tumor cells, a third label that binds a cell population determined to not be the tumor cells or a subset of the cell population, and a fourth label that selectively binds circulating tumor cells, wherein the first label is DAPI, the second label is Cytokeratin (CK), a key component of CTC, the third label is CD45, and the fourth label is human 5T4 antigen. The fluorescence sites of the slide are then scanned and analyzed by high performance calculations using algorithms that weight the cellular parameters detected by the first, second, third and fourth markers to identify and characterize circulating tumor cells.

The methods also provide methods for studying the prevalence of CTCs in 5T 4-positive cancer patients using fluorescence microscopy and cell attachment protocols. Another advantage of the method enables a pathologist to reposition and examine cells of interest for pathology confirmation and characterization. In the present invention, the protocol also includes removing the coverslip and/or lysing the water soluble smear medium on each of the fluorescently stained slides and re-staining the same cells with a second cell marker, such as standard giemsa switzerland, to provide additional insight into CTC morphology, size, and heterogeneity. Known CK's localized with high performance computational and cell attachment protocols can be morphologically evaluated⁺Individual rare cells and rare cell clusters. Although the fluorescence image of CTCs facilitates their proven identification, giemsa rapi dye provides additional cytological information about CTCs. In another aspect of the invention, the methods can be used to assess different cellular markers specific for a disease state, cell type, or cell state.

The ability to detect and characterize CTCs potentially facilitates diagnostic and personalized treatment of 5T 4-positive cancer patients. Due to their rarity, specialized methods are required to study CTCs. The present invention provides a fluid phase biopsy method that enables the use of standard cytopathological methods for detailed morphological characterization of CTCs in blood obtained from cancer patients and provides details of the cytological characteristics of a range of CTCs without the use of surface protein-based enrichment. Nucleated cells recovered from whole blood were placed on adhesive slides, immunofluorescent labeled, and 5T4 positive CTCs were analyzed by digital microscopy. Combining these techniques with conventional staining methods makes it possible to identify and evaluate CTCs using optical microscopy. Cells were observed using conventional pathology methods, CTCs exhibited a high degree of inter-and intra-patient polymorphism in whole blood preparations, and high and low nucleoplasmic ratios were used to identify intact CTCs and CTCs exhibiting markers of apoptosis. Morphological observations suggest that the full spectrum of (full spectrum) cells present in the primary tumor site and the metastatic tumor site can also be seen circulating in the blood and further provide a possible morphological classification framework to study the properties of subsets of cells associated with metastasis.

Automatic digital microscope

The coordinates of the expected cells are input into a rare-event imaging system (REIS), a fully automated scanning digital microscope system. The hardware components and proprietary scanning software of REIS are described in detail in other literature (Krivacic et al, proc. natl. acad. sci. usa 101:10501-10504, 2004).

Measuring

The detected fluorescent targets were analyzed by a software filtering operation to distinguish rare cells from false positive cells. Since cells are typically smaller than the laser spot resolution (20 μm), the first filter passes all targets below the threshold size (20 μm). The second filter analyzes the ratio between the fluorescence intensities of the different channels to eliminate homogeneous dye aggregates, which are common artifacts of immunofluorescent staining.

The sample may be prepared as a biological monolayer by aspirating a biological fluid sample, including but not limited to blood or a portion of blood from an individual. In one aspect, the sample is a monolayer of cells. Fluid samples are treated with fluorescent materials, such as, but not limited to, labeled dyes, that selectively bind different types of biomolecules (e.g., proteins, nucleic acids, or other molecules) that can be located on the surface of or within the cell. Suitable markers for marking a number of different cell types of clinical interest are known in the art, including selected cancer cell types, fetal cells, or other suitable cells of interest. In particular, markers for many other cells, such as brain cells, liver cells and bacterial cells, can be developed. The material emits a characteristic output, such as fluorescence or phosphorescence, in response to selected excitation radiation, such as by selected wavelength or spectral radiation, X-ray radiation, electron-beam radiation, and the like. Characteristic luminescence typically has a characteristic wavelength or spectral range of wavelengths. Although dyes are the primary labeling method, other techniques exist, including the use of labels known as quantum dots and DNA nanoparticle probes.

In another aspect of the invention, methods are provided for obtaining the location of rare cells, such as 5T4 positive Circulating Tumor Cells (CTCs), in a biological monolayer. See, for example, U.S. patent application No. 2004/0131241, which is incorporated herein by reference. A slide carrying at least one rare cell and having a cross-tag arranged at a position substantially forming a right angle is positioned on a slide holder of a first imaging system. A first coordinate space of the imaging system is defined and coordinates of the cross vane in the first coordinate space are specified. A second coordinate space of the second imaging system is defined and coordinates of the cross vane in the second coordinate space are specified. And calculating coordinate transformation parameters by using the coordinates specified by the cross measuring mark in the first coordinate space. Thereafter, coordinates of at least one object in a first coordinate space are specified, and the first coordinate space coordinates of the object are converted into unique coordinates in a second coordinate space using coordinate conversion parameters.

Once the rare cells or CTCs are located, the cover slips of the biological monolayer can be removed, or the water-soluble coverslipping tablets on each fluorescently stained slide can be dissolved. The same cells can be re-stained with a second cell marker, such as standard giemsa reyi dye, to gain insight into CTC morphology, size, and heterogeneity. Known cytokeratin positivity (CK) can be located⁺) Individual rare cells and rare cell clusters, and morphologically evaluated. Although the fluorescent image of CTCs facilitates their proven identification, giemsa rapi dye provides additional information about CTCs.

In another aspect, the method is used to assess different cellular markers characteristic of a disease, disease state, cell type, or cell state. The methods of the invention will facilitate the characterization of CTCs. It can confirm CTCs of blood obtained from 5T4 positive cancer patients in high quality without the need for enrichment and give insight into the morphology and characteristics of CTCs.

Studies of rare metastatic CTCs suggest that many CTCs are apoptotic and unable to form metastases, and that only one disseminated cancer cell out of 10,000 disseminated cancer cells is estimated to be able to establish metastases in fact. Thus, detection, morphological classification, and molecular characterization of these rare cells may target new and direct therapies, thus demonstrating the clinical significance of CTCs.

Cancer treatment

The method of cancer treatment is immunotherapy, in which the 5T4 antigen-specific antibody may be conjugated to a suitable drug, such as a cytotoxic agent or cytostatic agent, immunosuppressive agent, radioisotope, toxin, and the like. Antibody Drug Conjugates (ADCs) can be used to deliver drugs to 5T4 positive tumor cells or cancer cells in patients. ADCs for use in the treatment of 5T 4-positive cancers have been disclosed in U.S. patent No.8,309,094, which is incorporated herein by reference. Examples of ADCS are 5T 4-A1-mcMAF, 5T 4-A1-vcMAE and 5T4-vc-MMAD, where 5T4-A1 is a humanized antibody that specifically binds to the 5T4 antigen and MMAE, MMAE and MMAD are Auristatin derivatives. Auristatin has been shown to interfere with microtubule dynamics as well as nuclear division and cell division, and has anti-cancer activity.

Diagnostic assay

Figure 1a illustrates an embodiment of the invention in which quantification of the 5T4 antigen on CTCs is used to generate an "H score" which is summed by multiplying the percentage of CTCs within each category by their respective category value to yield a score of 0-300. As shown in fig. 1b, the scoring system utilized 5T4 expression as determined by an optimized 5T44 color analysis, the 5T44 color analysis being described in example 1, using a panel of NSCLC cell lines selected based on 5T4 expression levels. These levels were confirmed by standard immune cell (ICC) staining experiments. These cell lines represent high (MDA-MB 435 and NCI-H226 cell lines), medium (NCI-H1975 and MDA-MB 361 cell lines) and low (NCI-H522 and NCI-H2122 cell lines) expression levels of 5T 4. The average expression level of 5T4 in each of these cell lines was used to establish thresholds for high, medium and low expression of 5T4 in the assay.

In another embodiment of the invention, cancer patients are screened for the presence of CTCs expressing the 5T4 antigen using the optimized 5T44 color diagnostic assay described in example 1. This analysis helped determine the expression level of the 5T4 antigen on CTCs by calculating and characterizing CTCs, and determining the correlation between expression of the CTC 5T4 target and expression of 5T4 in primary tumors. The 5T4 expression scatter plot depicted in fig. 2a is for single CTCs and CTC clusters calibrated with control cell lines. As shown in 2b, 17 non-small cell lung cancer (NSCLC) patient samples were treated with an optimized 5T44 color analysis. Therefore, the H-score category is determined using the 5T4-4 color analysis of the present invention and then used for H-score calculation. Finally, a diagnostic assay to identify CTCs expressing a 5T4 target would be used to identify a treatable cancer patient population and serve as a means to monitor CTCs in cancer patients during treatment with ADCs such as 5T4-a 1-mcMMAF.

Another aspect of the invention is the use of H-scores as a basic scoring system to characterize 5T4 on CTCs. As indicated above, the H-score is a weighted score that multiplies the percentage of CTCs within each category by their respective category values and then sums, resulting in a score of 0-300. As shown in fig. 1a, CTCs were classified into 4 classes (0-3) based on individual 5T4 expression. In order to calculate a reasonably usable H-score, a minimum of 10 CTCs must be present.

For the patient, the H-score will be calculated in two ways: (1) tradition H-score (THS) -count the average 5T4 intensity for each event (single CTC or CTC cluster) as a single data point; (2) cluster weighted H-score (CWHS) — (within a single CTC or cluster) the average 5T4 intensity for each CTC was counted as a single data point.

Using the values of the H-score categories and the percentage of CTCs for each category, an example of calculating an H-score is as follows: h score category 0 (1.5% CTCs); h score category 1 (15.0% CTCs); h score category 2 (68% CTCs); h scored category 3 (15.5% CTCs). H score (1.5 × 0) + (15.0 × 2) + (68.0 × 2) + (15.5 × 3) ═ 198.

The H-score can then be used to select the patient population with the greatest probability of successful treatment with an ADC such as 5T4-a1-mcMMAF or other 5T4 specific ADC. Figure 2b provides H scores for 14 of 17 NSCLC patients calculated using the 5T44 color analysis of the present invention.

In other embodiments, methods of treating cancer are provided, comprising identifying a patient having a 5T 4-positive cancer by identifying 5T 4-positive CTCs with an optimized 5T44 color analysis; classifying the patient by determining an H-score; and administering to a patient in need thereof an effective amount of an ADC that specifically binds to a 5T4 positive cancer. Moreover, patients were intermittently monitored for the presence of 5T4 positive CTCs using an optimized 5T44 color analysis during treatment. The detection of a decrease in the number of 5T4 positive circulating tumor cells in the blood sample after treatment with the ADC, as compared to the number of 5T4 positive circulating tumor cells in the blood sample before treatment with the ADC, may indicate the efficacy of the antibody drug conjugate compound in treating 5T4 positive cancer in a mammalian subject.

In another embodiment, the cell population is analyzed using an optimized 5T44 color analysis to identify and characterize circulating tumor cells in the test sample prior to treatment with the antibody drug conjugate as compared to after treatment with the antibody drug conjugate, wherein a change in the ratio of 5T4 positive circulating tumor cells to 5T4 negative circulating tumor cells in the blood sample after treatment as compared to the ratio of 5T4 positive circulating tumor cells to 5T4 negative circulating tumor cells in the blood sample before treatment may indicate efficacy of the antibody drug conjugate in reducing 5T4 positive circulating tumor cells.

In some embodiments, a method of treating cancer comprises identifying a patient having a 5T 4-positive cancer by identifying 5T 4-positive CTCs with optimized 5T44 color analysis, and administering to the patient an effective amount of ADC and a chemotherapeutic agent that specifically binds to 5T 4-positive cancer. Chemotherapeutic agents are drugs that have not been found to be refractory to the treatment of cancer. In some embodiments, the chemotherapeutic agent is an agent with which it has been found to be refractory to the treatment of cancer. The ADC may be administered to a patient who has undergone therapy (e.g., surgery to treat cancer). In another embodiment, the other treatment method is radiation therapy. Moreover, patients were intermittently monitored for the presence of 5T4 positive CTCs using an optimized 5T44 color analysis during treatment.

Detectable label

The specific label or detectable group used in the assay can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The particular type of label is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody to the cell marker on the cell or circulating tumor cell used in the assay. The detectable group may be any substance having a detectable physical or chemical property. Such detectable labels have been well developed in the field of assays or immunoassays, and in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention includeFluorescent dyes (Invitrogen), magnetic beads (e.g., dynabeads. tm.), fluorescent dyes (e.g., fluorescein isothiocyanate, deksa.)Red (Texas red), rhodamine, etc.), radioactive labels, and other imaging agents such as microbubbles (for ultrasound imaging), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As noted above, a variety of labels can be used, the choice of label depending on the sensitivity desired, ease of conjugation with the compound, stability requirements, available instrumentation, and processing specifications.

The non-radioactive label is often attached by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand is then bound to an anti-ligand molecule (e.g., streptavidin) that is either inherently detectable or covalently bound to a signaling system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. Many ligands and anti-ligands can be used. If the ligand has a natural anti-ligand, such as biotin, thyroxine and cortisol, it can be used in conjunction with a labeled naturally occurring anti-ligand. Alternatively, any hapten or antigenic compound can be used in combination with the antibody.

The molecule may also be conjugated directly to a signal generating compound, for example by conjugation to an enzyme or fluorophore. The enzymes of interest as tags are primarily hydrolases, in particular phosphatases, esterases and glycosidases, or oxidoreductases, in particular peroxidases. The fluorescent compound comprisesFluorescent dyes (Invitrogen), fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds include luciferin and 2, 3-dihydrophalazenediones, such as luminol. For a review of the various tagging or signal generating systems that may be used, see U.S. patent No. 4,391,904, which is incorporated herein by reference.

Methods for detecting tags are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, the means of detection includes scintillation counting or a photosensitive film in autoradiography. If the label is a fluorescent label, it can be detected by exciting a fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. The fluorescence may be detected visually through a photosensitive film, by using an electron detector such as a Charge Coupled Device (CCD) or a photomultiplier tube, or the like. Similarly, enzyme labels may be detected by providing the enzyme with a suitable substrate and detecting the resulting reaction product. Finally, simple calorimetric labels can be detected simply by observing the color associated with the label.

Other embodiments and uses will be apparent to those skilled in the art in light of this disclosure.

Example 1

Optimized 5T44 color diagnostic analysis

An optimized 4-channel assay was developed to identify 5T4 positive CTCs. CTCs were stained with 4 different dyes and measured on 4 independent channels. For example, CTC may be used against CK-(Red) anti(Green), anti-5T 4-(purple) staining; while nuclei can be stained blue with DAPI. Because of multiple coloursFluorescent dyes (Invitrogen) can be used, so other combinations of staining can be selected.

Patient and blood sample collection

Samples were collected from metastatic cancer patients in anticoagulated tubes and processed within 24 hours. Blood samples were also drawn from normal controls.

Blood sample processing for CTC detection

The blood samples were shaken for 5 minutes before White Blood Cell (WBC) counts were measured using the Hemocue WBC system (Hemocue, Sweden). Based on WBC counts, a volume of blood was subjected to red cell lysis (ammonium chloride solution). After centrifugation, nucleated cells were resuspended in PBS and attached as a monolayer onto a custom-made glass slide. The glass slide is the same size as a standard microscope slide, but has a proprietary coating that allows for maximum retention of living cells. Each slide contains about three million nucleated cells; the number of cells seeded on each slide is therefore dependent on the WBC count of the patient.

For the present study, 4 slides were used as the test in order to detect CTCs in cancer patients. The remaining slides generated for each patient were stored at-80 ℃ for future experiments. 4 slides from each patient were thawed, and cells were fixed with 2% paraformaldehyde, permeabilized with cold methanol, and non-specific binding sites blocked with goat serum. Monoclonal anti-Broadspectrum cytokeratin antibody (Sigma) and CD45-Alexa fluorescent dye (Serotec) were then incubated with the slides for 40 minutes at 37 ℃. After PBS washing, goat anti-mouse antibody-Alexa fluorescent dye (Invitrogen) was incubated with the slides for 20 min at 37 ℃. After PBS washing, anti-5T 4 antibody-Alexa fluorescent dye was subsequently incubated with the slide for 20 minutes at 37 ℃. Cells were counterstained with DAPI for 10 min and mounted with water-sealed tablets.

Imaging and technical analysis

All 4 slides per patient were scanned with a custom fluorescence scanning microscope developed and optimized for rapid and reliable scanning. Each slide was scanned completely in 4 colors at 10 x magnification and produced over 6900 images. The resulting images are provided to an analysis algorithm that identifies potential candidate CTCs based on a number of measurements including cytokeratin intensity, CD45 intensity, 5T4 intensity, and shape and size of the nucleus and cytoplasm. The technical analyst then scrutinizes the algorithm, producing potential candidates, and removes points (hit) that are apparently not cells, such as dye aggregates.

Professional analysis and interpretation

All possible candidate CTCs are presented to the hematopathologist for analysis and interpretation via a web-based report, where the hematopathologist lists each candidate cell as a CTC or exclusion. Cells are classified as CTCs if they are cytokeratin positive, 5T4 positive, CD45 negative, contain intact DAPI nuclei without identifiable apoptotic changes (blebbing, degenerate appearance) or disrupted appearance, and are morphologically distinct from the surrounding WBCs. The cell must have a cytoplasm that is apparently around and contains an intact nucleus. The cytoplasm may exhibit apoptotic changes, such as blebbing and density irregularities, or slight disruption of the peripheral cytoplasmic borders, but it may not be disrupted so that its association with the nucleus is problematic. The image is presented as a digital image, which can be displayed by a single fluorescent channel as well as a composite image. Each cell image was annotated with auxiliary statistical data regarding relative nuclear size, fluorescence intensity, and comparable fluorescence intensity. Each CTC candidate is presented in the field of view with sufficient surrounding WBCs to allow a contextual comparison (contextual composition) between the cytomorphological characteristics of the cell in question and the background WBCs.

Staining with Giemsa rapae

The coverslip was removed from the fluorescent stained slide and rinsed in PBS. The slides were then submerged for 3 minutes with Fisher Scientific, Kalamazoo, Mich. To the dye covered slide, 1.5 mL of phosphate buffer, pH 6.8 (Fisher Scientific, kalamazo, Mich.) was added, and the dye and buffer were mixed together by gently shaking for 1 minute. The mixture was then allowed to rest on the slide for more than 2 minutes, after which the slide was rinsed with deionized water and allowed to air dry.

The steps used in the CTC identification and characterization method of the optimized 5T44 color analysis of the present invention were: (1) preparing a glass slide; (2) storing the glass slide; (3) thawing and staining the glass slide; (4) scanning the glass slide; (5) running an algorithm; and (6) technical analysis and reporting.

CTC analysis was specifically developed in view of clinical environment as well as early technological innovation and future automation. All experimental procedures followed strict standard operating procedures that had been optimized, tested and validated. Data collection and candidate identification have been automated with a specific interface that enables a pathologist to make decisions and then track those decisions.

This system promise enables new research to perform molecular characterization of CTC morphological classifications and to be applied to on-site, point-of-care screening, monitoring and management of cancer patients.

Claims

1. Use of an anti-5T 4 antibody in the preparation of a diagnostic agent for detecting 5T 4-positive circulating tumor cells in a mammalian subject suspected of having a 5T 4-positive cancer, by a method comprising the steps of:

testing a blood sample from the mammalian subject, wherein the blood sample comprises a population of cells;

placing the blood sample on a substrate;

detecting the presence or absence of nucleated cells in the blood sample using a first label;

detecting the presence or absence of expression of a second marker on cells in the blood sample, wherein the second marker is cytokeratin;

detecting the presence or absence of expression of a third marker on cells in the blood sample, wherein the third marker is CD 45;

detecting the presence or absence of expression of a fourth marker on cells in the blood sample, wherein the fourth marker is human 5T4 antigen; and

analyzing the first, second, third, and fourth markers in the population of cells to identify and characterize the circulating tumor cells.

2. The use of claim 1, wherein detection of 5T 4-positive circulating tumor cells in the blood sample indicates the presence of an early stage 5T 4-positive cancer in the mammalian subject.

3. The use of claim 1, wherein detection of the absence of 5T 4-positive circulating tumor cells in the blood sample indicates that the mammalian subject is in a disease-free state or a non-measurable disease state, wherein the disease is 5T 4-positive cancer.

4. The use of claim 1, wherein detection of the presence or absence of 5T 4-positive circulating tumor cells in the blood sample is indicative of 5T 4-positive cancer treatment or treatment management during cancer recovery.

5. The use of claim 1, wherein said cell population is a mixed cell population.

6. The use of claim 1, wherein the substrate is a planar substrate.

7. The use of claim 1, wherein the substrate is a microfluidic device.

8. The use of claim 1, wherein the substrate is a cassette containing an enriched cell population.

9. The use of claim 1, wherein the sample is placed on the substrate to form a biological monolayer.

10. The use of claim 1, wherein the first label, the second label, the third label, or the fourth label is detected using fluorescence.

11. The use of claim 1, wherein said first marker is used to analyze said population of cells by nuclear details, nuclear contour, presence or absence of nucleoli, cytoplasmic character or amount of cytoplasm.

12. The use of claim 11, wherein the first marker is DAPI.

13. The use of claim 11, wherein said method further comprises analyzing said cell population by measuring intact cells with high nucleoplasmic ratio, intact cells with low nucleoplasmic ratio, early apoptotic cells, or late apoptotic cells, and identifying said circulating tumor cells and circulating tumor cell clusters.

14. The use of claim 1, wherein said method further comprises cell staining to identify said circulating tumor cells by morphology, size, or nuclear to cytoplasmic ratio.

15. The use of claim 14, wherein the cell staining is performed using giemsa rapi dye.

16. The use of claim 1, wherein said method further comprises analyzing said population of cells by their attachment to said substrate, scanning said population of cells on said substrate, and imaging said cells by digital microscopy with repositioning.

17. The use of claim 1, wherein the method further comprises quantifying human 5T4 antigen on the circulating tumor cells to produce an H-score, wherein the H-score is used to select a population of 5T 4-positive cancer patients.

18. The use of claim 1, wherein the detection of the presence of the first marker, the presence of the second marker, the absence of the third marker, and the presence of the fourth marker indicates the presence of 5T4 positive circulating tumor cells.

19. The use of claim 1, wherein detection of the presence of 5T4 positive circulating tumor cells in the blood sample indicates the presence of a 5T4 positive cancer in the mammalian subject.

20. The use of claim 19, wherein the 5T 4-positive cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, and testicular cancer.

21. Use of an anti-5T 4 antibody for the preparation of a diagnostic agent for diagnosing a 5T 4-positive cancer in a mammalian subject suspected of having a 5T 4-positive cancer, by a method comprising the steps of:

placing the blood sample on a substrate;

analyzing and quantifying the first, second, third, and fourth markers in the cell population to identify and characterize circulating tumor cells.

22. The use of claim 21, wherein quantifying human 5T4 antigen on the circulating tumor cells is used to generate an H-score, wherein the H-score is used to select a population of 5T4 positive cancer patients.

23. The use of claim 21, wherein the first marker is DAPI.

24. The use of claim 21, wherein the detection of the presence of the first marker, the presence of the second marker, the absence of the third marker, and the presence of the fourth marker indicates the presence of 5T4 positive circulating tumor cells.

25. Use of an anti-5T 4 antibody in the manufacture of a diagnostic agent for screening for the activity or efficacy of an antibody drug conjugate for treating a 5T4 positive cancer in a mammalian subject suspected of having cancer, by a method comprising the steps of:

administering to the mammalian subject a therapeutically effective amount of an antibody drug conjugate;

placing the blood sample on a substrate;

analyzing the first, second, third, and fourth labels in the cell population to identify and characterize circulating tumor cells in the blood sample before treatment with the antibody drug conjugate compared to after treatment with the antibody drug conjugate,

wherein a change in the ratio of 5T4 positive circulating tumor cells to 5T4 negative circulating tumor cells in the blood sample after treatment as compared to the ratio of 5T4 positive circulating tumor cells to 5T4 negative circulating tumor cells in the blood sample before treatment may indicate the efficacy of the antibody drug conjugate in reducing 5T4 positive circulating tumor cells.

26. The use of claim 25, further comprising quantifying human 5T4 antigen on the circulating tumor cells to produce an H-score, wherein the H-score is used to select a population of 5T4 positive cancer patients.

27. The use of claim 25, wherein the first marker is DAPI.

28. The use of claim 25, wherein the detection of the presence of the first marker, the presence of the second marker, the absence of the third marker, and the presence of the fourth marker indicates the presence of 5T4 positive circulating tumor cells.

29. The use of claim 25, wherein the antibody drug conjugate is anti-5T 4-a 1-mcMMAF.

30. The use of claim 17 or 21, wherein the first, second, third and fourth markers are analyzed and quantified for determining the inclusion or exclusion of a mammalian subject from treatment for a 5T 4-positive cancer.

31. A diagnostic agent for detecting 5T4 positive circulating tumor cells in a mammalian subject suspected of having a 5T4 positive cancer, comprising:

a first label that binds to nucleated cells in a blood sample from the mammalian subject, the blood sample being disposed on a substrate, wherein the blood sample comprises a population of cells;

an antibody for detecting the presence or absence of expression of a second marker on cells in the blood sample, wherein the second marker is cytokeratin;

an antibody for detecting the presence or absence of expression of a third marker on cells in the blood sample, wherein the third marker is CD 45; and

an anti-5T 4 antibody for detecting the presence or absence of expression of a fourth marker on cells in the blood sample, wherein the fourth marker is human 5T4 antigen;

wherein the first, second, third and fourth markers in the population of cells are analyzed to identify and characterize the circulating tumor cells.

32. The diagnostic agent of claim 31, wherein detection of 5T 4-positive circulating tumor cells in the blood sample indicates the presence of an early stage 5T 4-positive cancer in the mammalian subject.

33. The diagnostic agent of claim 31, wherein detection of the absence of 5T 4-positive circulating tumor cells in the blood sample indicates that the mammalian subject is in a disease-free state or a non-measurable disease state, wherein the disease is 5T 4-positive cancer.

34. The diagnostic agent of claim 31, wherein detection of the presence or absence of 5T 4-positive circulating tumor cells in the blood sample is indicative of 5T 4-positive cancer treatment or treatment management during cancer recovery.

35. The diagnostic agent of claim 31, wherein said cell population is a mixed cell population.

36. The diagnostic agent of claim 31, wherein said matrix is a planar matrix.

37. A diagnostic agent according to claim 31 wherein the substrate is a microfluidic device.

38. The diagnostic agent of claim 31, wherein the matrix is a cassette that holds an enriched cell population.

39. A diagnostic agent as claimed in claim 31 wherein the sample is placed on the substrate to form a biological monolayer.

40. The diagnostic agent of claim 31, wherein said first label, said second label, said third label, or said fourth label is detected using fluorescence.

41. A diagnostic agent as claimed in claim 31 wherein said first marker is used to analyse said population of cells by nuclear details, nuclear contours, the presence or absence of nucleoli, cytoplasmic traits or the amount of cytoplasm.

42. The diagnostic agent of claim 41, wherein said first marker is DAPI.

43. The diagnostic agent of claim 41, wherein analyzing said cell population further comprises measuring intact cells with a high nuclear to cytoplasmic ratio, intact cells with a low nuclear to cytoplasmic ratio, early apoptotic cells, or late apoptotic cells analyzing said cell population and identifying said circulating tumor cells and circulating tumor cell clusters.

44. The diagnostic agent of claim 31, wherein said analysis further comprises cell staining to identify said circulating tumor cells by morphology, size or nuclear to cytoplasmic ratio.

45. The diagnostic agent of claim 44, wherein the cell staining is performed using a Giemsa rapae dye.

46. The diagnostic agent of claim 31, wherein analyzing said cell population further comprises analyzing attachment of cells to said substrate, scanning said cell population on said substrate, and imaging said cells with repositioning by digital microscopy.

47. The diagnostic agent of claim 31, wherein analyzing the cell population further comprises quantifying human 5T4 antigen on the circulating tumor cells to produce an H-score, wherein the H-score is used to select a population of 5T 4-positive cancer patients.

48. The diagnostic agent of claim 31, wherein detection of the presence of 5T4 positive circulating tumor cells in the blood sample indicates the presence of a 5T4 positive cancer in the mammalian subject.

49. The diagnostic agent of claim 31, wherein the 5T 4-positive cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, stomach cancer, and testicular cancer.

50. The diagnostic agent of claim 31, wherein detection of the presence of the first marker, the presence of the second marker, the absence of the third marker, and the presence of the fourth marker indicates the presence of 5T4 positive circulating tumor cells.

51. The diagnostic agent of claim 31, wherein the presence of the first marker, the presence of the second marker, the absence of the third marker, and the presence of the fourth marker are detected for determining inclusion of a mammalian individual in a treatment for a 5T 4-positive cancer.