Methods for detecting cancer in a subject

Abstract

Provided herein are methods of detecting a cancer in a subject, the method comprising (a) obtaining a biological sample from the subject, wherein the biological sample comprises a plurality of cells; (b) enriching a target cell from the plurality of cells; and (c) analyzing the target cell and quantifying one or more features of the target cell, thereby detecting the cancer in the subject.

Description

[0001] METHODS FOR DETECTING CANCER IN A SUBJECT

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority’ to U.S. Provisional Patent Application No. 63 / 542,935, filed on October 6, 2023, which is incorporated herein by reference in its entirety7.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant CA276704 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to the field of cancer detection. In particular, it relates to detecting a cancer cell from a biological sample (e.g., body fluids) from a subject by using cell enrichment and / or flow cytometry.

[0008] BACKGROUND

[0009] Cancer is a local disease at an early stage, which then systemically evolves as it progresses by triggering alterations in surrounding microenvironment, disturbing immune surveillance and further disseminating its molecular contents into circulation. This pathogenic characteristic of cancer makes the use of body fluids from a subject (e.g., blood / serum / plasma, urine, tears, and cerebrospinal fluids) credible surrogates harboring tumor tissue-derived molecular alterations for the detection of cancer. However, despite being the second leading cause of death worldwide, many cancers do not have screening programs and many people with a high risk of developing cancer fail to follow the advised medical screening regime due to the nature of the available screening tests and other challenges with compliance.

[0010] Cancer biomarkers are measurable indicators that play vital roles in clinical applications, wherein biomarkers in body fluids have gained considerable attention since the development of liquid biopsy, and their data volume is rapidly increasing. Biomarkers are biochemical, cellular, gene-related, or molecular indicators of diseases that are measurable in physical media, such as blood, body fluids, tissues, or cells. These biomarkers can be widely used in clinical applications including early cancer diagnosis, improved cancer prognosis, early detection of cancer relapse, real-time monitoring of therapeutic efficacy and therapeutic resistance. SUMMARY

[0011] Provided herein are methods of detecting a cancer in a subject, the method comprising (a) obtaining a biological sample from the subject, wherein the biological sample comprises a plurality of cells; (b) enriching a target cell from the plurality of cells; and (c) analyzing the target cell and quantifying one or more features of the target cell, thereby detecting the cancer in the subject. In some embodiments, the method further comprises dissociating the plurality of cells into single cells before the enrichment step (b). In some embodiments, the dissociating and enrichment step comprises using an alcohol-based cytology preservative. In some embodiments, the enrichment step (b) comprises separating the target cell based on the target cell size. In some embodiments, the enrichment step (b) comprises enriching the target cell based on the target cell susceptibility to lysis.

[0012] In some embodiments, the one or more features comprise a presence of aneuploidy, global hypomethylation, tumor suppressor protein loss, presence of cancer specific biomarkers, phosphorylation of oncoproteins, elevated proliferation rate, tetraploidy, elevated nuclear to cytoplasmic ratio, or any combination thereof. In some embodiments, the one or more features comprise the presence of aneuploidy in the target cell. In some embodiments, the one or more features comprises global hypomethylation of DNA in the target cell.

[0013] In some embodiments, the analyzing step (c) comprises flow cytometry. In some embodiments, the analyzing step (c) comprises staining the target cell with a fluorescent dye and / or an antibody. In some embodiments, the fluorescent dye comprises GelRed, SYBR green, DAPI, Propidium Iodide, or fluorescein. In some embodiments, the fluorescent dye is conjugated to the antibody, wherein the antibody specifically binds to methylated DNA, a tumor suppressor protein, or a phosphoryl-group on an oncoprotein. In some embodiments, the antibody is an anti-CAM 5.2 antibody, an anti-cytokeratin antibody, an anti-CD45 antibody, an anti-CD66b antibody, an estrogen receptor antibody, or a progesterone receptor antibody.

[0014] In some embodiments, the biological sample comprises saliva, pap test fluid, urine, cerebrospinal fluid, ascites fluid, or pleural fluid from the subject. In some embodiments, the biological sample comprises urine from the subject. In some embodiments, the target cell comprises a urothelial cell. In some embodiments, the biological sample comprises pap test fluid from the subject. In some embodiments, the target cell comprises an endometrial cell.

[0015] Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0017] BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 shows an exemplary schematic of a bladder cancer detection strategy for the enrichment and analysis of urothelial tumor cells from a voided urine sample. Flow cytometry graphs show hypothetical sample containing normal (blue) and cancerous (red) urothelial cells.

[0019] FIG. 2 shows diameter of tumor and normal cells in saliva, urine, and pap smears. (FIG. 2A) shows p!6 immunohistochemistry on representative saliva specimens from patients with head and neck squamous cell carcinoma illustrating the morphology of tumor cells shed into saliva. (FIG. 2B) shows HPV PCR results compared to total DNA content across different size fractions of saliva from tw o patients with head and neck squamous cell carcinoma. (FIGs. 2C- 2E) show cell size distributions of tumor-initiating (e.g., urothelial, endometrial) or tumor (e.g., pl 6+ in saliva) cells compared to the main normal cell components of urine, pap smears, and saliva as assessed by microscope.

[0020] FIG. 3 shows leukocyte depletion across urine, pap smears, and saliva. Leukocytes are removed by washing samples over a 5 pm filter with a chaotropic agent, leaving a pure population of epithelial cells on top of the filter. Samples were stained by SYBR green and SYPRO orange before imaging under a fluorescence microscope. Arrows indicate urothelial carcinoma cells against a background of dense leukocytes.

[0021] FIG. 4 shows enrichment of tumor cells from pure tissue fragments in pap smear samples, as demonstrated by the recovery’ of aneuploidy in the 5-20 pm size fraction after mechanical tissue dissociation. (FIG. 4A, FIG. 4B) show pattern of aneuploidy in Tao brush samples (uterine scrapings) from two patients with endometrioid and serous uterine carcinomas, respectively. (FIGs. 4C-4J) show’ mixtures of the Tao samples in A and B, respectively, each with a different normal pap smear sample. Fraction above the 20 pm filter (FIG. 4C, FIG. 4D) and between the 5 and 20 pm filters (FIG. 4E, FIG. 4F) without prior mechanical cell dissociation. Fraction between the 5 and 20 pm, filters, (FIG. 4G, FIG. 4H) with prior mechanical cell dissociation and (FIG. 41, FIG. 4J) with prior dissociation and washing of the 5 pm filter with a chaotropic agent.

[0022] FIG. 5A shows evaluation of an enrichment workflow by sequencing. TERT hotspot mutant allele fractions before and after passage through workflow. FIG. 5B shows optimization of squamous cell depletion. In the subset of samples from panel A with >80% squamous cells, a subsequent experiment showed a workflow with two rounds of 15 pm filtration enhanced enrichment over a workflow with one round of 15 pm filtration.

[0023] FIG. 6 shows how urothelial cancer cells can be detected in a human urine sample through selective gating of cytokeratin positive cells to reveal a population with aneuploid DNA content.

[0024] FIG. 7 shows how endometrial cancer cells can be detected in a human pap test sample through selective gating of cytokeratin positive cells to reveal a population with aneuploid DNA content (top right).

[0025] FIG. 8 shows validation of 5mC antibody stain on cancer cell line HCT116. The cell line labeled DKO has had its DNA methyltransferases knocked out and has been shown to demonstrate global hypomethylation.

[0026] FIG. 9 shows validation of PTEN antibody staining for flow cytometry on SurePath- preserved cell lines, one of which is wild-ty pe for PTEN and the other of which has had PTEN knocked out.

[0027] FIG. 10 shows that CAM5.2 still selectively stains urothelial cells (T24 cells) after exposure to the HC1 required to co-stain with an antibody against 5 -methyl cytosine.

[0028] FIG. 11 shows controls showing diploid DNA content.

[0029] FIG. 12 shows several of the cases showing CAM5.2 positive cell populations with clear aneuploidy (labeled ’A").

[0030] FIG. 13 shows that controls do not show distinct populations of CAM5.2 positive cells that are hypomethylated.

[0031] FIG. 14 shows that several of the cases show distinct populations of CAM5.2 positive cells that have a global reduction in methylation (labeled “H”). DETAILED DESCRIPTION

[0032] Cancer is a complex disease with a high mortality rate and is a major global public health concern, and cancer biomarkers have been widely used for cancer diagnosis and treatment since their development. The identification of circulating biomarkers in body fluids (e.g., urine, blood, saliva, body-cavity fluids) can be used in cancer diagnostics by relatively easily obtaining biological samples using minimally invasive procedures before, during, and after cancer treatment. Additionally, the availability of various technologies which perform high-throughput and informative biomolecular analyses on limiting sample amounts is boosting biomarkers screening studies. Liquid biopsy shows promise for cancer screening and diagnostics.

[0033] Provided herein are methods of detecting a cancer in a subject that include (a) obtaining a biological sample from the subject, wherein the biological sample comprises a plurality of cells; (b) enriching a target cell from the plurality of cells; and (c) analyzing the target cell and quantifying one or more features of the target cell. In some embodiments, methods provided herein include enriching a target cell from the plurality of cells. In some embodiments, methods provided herein include analyzing a target cell and quantifying one or more features of the target cell. In some embodiments, methods provided herein include both enriching a target cell from the plurality of cells and analyzing a target cell and quantifying one or more features of the target cell.

[0034] Various non-limiting aspects of these methods are described herein and can be used in any combination without limitation. Additional aspects of various components of methods for detecting a cancer from a biological sample from a subject are known in the art.

[0035] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0036] As used herein, the term “about,” when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%. 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0037] As used herein, the term “biological sample” refers to a sample obtained from a subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. A biological sample can be obtained from a eukary ote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). The biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy. In some embodiments, biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory' disorders, metabolic disorders, nervous system disorders, and cancer.

[0038] The biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.

[0039] As used herein, biological samples can include but are not limited to plasma, serum, blood, tissue, tumor sample, stool, sputum, saliva, urine, sweat, tears, ascites, bronchoalveolar lavage, semen, archeologic specimens, Pap test samples, and forensic samples. In some embodiments, the biological sample is a solid biological sample (e.g., a tumor sample). In some embodiments, the biological sample is a liquid biological sample. Liquid biological samples can include, but are not limited to plasma, serum, blood, sputum, saliva, urine, sweat, tears, ascites, bronchoalveolar lavage, and semen. In some embodiments, the biological sample is a plasma or serum sample. In some embodiments, the liquid biological sample is a whole blood sample. In some embodiments, the liquid biological sample comprises peripheral mononuclear blood cells. In some embodiments, the biological sample is a cerebrospinal fluid (CSF) sample.

[0040] As used herein, the terms "‘cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma”, refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g.. benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0041] As used herein, the term ‘'subject” is intended to refer to any subject. In some embodiments, the subject is cat, a dog, a goat, a human, a non-human primate, a rodent (e.g., a mouse or a rat), a pig, or a sheep. In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0042] Method of Detecting a Cancer in a Subject

[0043] Provided herein are methods of detecting a cancer in a subject, a method including (a) obtaining a biological sample from the subject, wherein the biological sample comprises a plurality of cells; (b) enriching a target cell from the plurality of cells; and (c) analyzing the target cell and quantifying one or more features of the target cell, thereby detecting the cancer in the subject. In some embodiments, methods provided herein include enriching a target cell from the plurality of cells. In some embodiments, methods provided herein include analyzing a target cell and quantifying one or more features of the target cell. In some embodiments, methods provided herein include both enriching a target cell from the plurality of cells and analyzing a target cell and quantifying one or more features of the target cell. Obtaining a Biological Sample from a Subject

[0044] In some embodiments, the obtaining step (a) comprises collecting a biological sample (e.g., a blood sample, a urine sample) from the subject. In some embodiments, a biological sample can include but is not limited to plasma, serum, blood, tissue, tumor sample, stool, sputum, saliva, urine, sweat, tears, ascites, bronchoalveolar lavage, semen, archeologic specimens, and forensic samples. In some embodiments, a biological sample is a solid biological sample (e.g., a tumor sample). In some embodiments, a biological sample is a liquid biological sample. Liquid biological samples can include, but are not limited to plasma, serum, blood, sputum, saliva, urine, sweat, tears, ascites, bronchoalveolar lavage, and semen. In some embodiments, a biological sample is a plasma or serum sample. In some embodiments, a liquid biological sample is a whole blood sample. In some embodiments, a liquid biological sample comprises peripheral mononuclear blood cells. In some embodiments, a biological sample is a cerebrospinal fluid (CSF) sample. In some embodiments, a biological sample can include a body fluid. In some embodiments, a biological sample can be collected from a body fluid from a pap smear test. In some embodiments, a biological sample can be collected from urine. In some embodiments, a biological sample can be collected from saliva. In some embodiments, a biological sample can be collected from a body fluid into which cells are exfoliated. In some embodiments, a biological sample can include cerebrospinal fluid, ascites fluid, or pleural fluid. In some embodiments, a biological sample can be collected by using any sample collection method known in the art.

[0045] In some embodiments, a biological sample can include saliva, pap test fluid, urine, cerebrospinal fluid, ascites fluid, or pleural fluid from the subject. In some embodiments, a biological sample comprises urine from a subj ect, wherein a target cell in the biological sample comprises a urothelial cell. In some embodiments, a biological sample comprises pap test fluid from a subject, wherein a target cell in the biological sample comprises an endometrial cell.

[0046] Enriching a Target Cell

[0047] In some embodiments, methods provided herein include an enrichment step. In some embodiments, the plurality of cells is dissociated into one or more single cells prior to enrichment. Dissociating refers to any procedure that disaggregates a pl urality of cells that may be in clumps whilst not disrupting cellular integrity. In some embodiments, dissociating a plurality of cells includes enzymatically disaggregating the plurality of cells in the biological sample. In some embodiments, dissociating a plurality of cells includes mechanically disaggregating the plurality of cells in the biological sample. In some embodiments, dissociating a plurality of cells can include enzymatically and mechanically disaggregating the plurality of cells in the biological sample.

[0048] In some embodiments, the enrichment step comprises using an alcohol-based cytology preservative. In some embodiments, the enrichment step can be performed on a biological sample that has been stored in an alcohol-based cytology preservative. In some embodiments, an alcohol-based cytology’ preservative can include, but is not limited to, ThinPrep® or SurePath™ preservatives.

[0049] As used herein, the terms “enriching” or “enrichment” refer to increasing a proportion of a target cell type out of a biological sample that includes multiple cell ty pes. Cell enrichment methods are used to increase a proportion of one or more target cells of interest out of a biological sample composed of a mix of different cell types. Enrichment of target cells enables performance of various types of analysis, including without limitation enumeration, phenotypic characterization, molecular analysis, microscopic and digital image analysis, or further cell sorting of the subpopulation of cells. In some embodiments, enrichment of target cells can include antibody-based enrichment. In some embodiments, antibody -based enrichment can include enriching cells based on expression of proteins that are targeted by antibodies. In some embodiments, enrichment of target cells can include enrichment based on density properties of the target cells. In some embodiments, a method for density-based enrichment can include using density gradient.

[0050] In some embodiments, the enrichment step (b) comprises separating the target cell based on the target cell size. In some embodiments, a method for size-based cell enrichment can include membrane microfiltration, pinched flow fractionation, deterministic lateral displacement, or hydrophoresis. In some embodiments, “cell size” refers to the dimensions of the cell. In some embodiments, a target cell may be approximately spherical, and the cell size refers to the approximate diameter of the cell. In some embodiments, a target cell may be non- spherical, wherein size-based enrichment is based on the smallest diameter of the cell.

[0051] In some embodiments, the enrichment step (b) comprises enriching a target cell based on the target cell susceptibility to lysis. In some embodiments, separation comprises separating a target cell based on differences in the susceptibility to lysis of the target cell compared to a non-target cell. For example, a biological sample may be washed with a solution that lyses nontarget cells but not the target cells, thereby allowing the target cells to be enriched. In some embodiments, a lysis solution can include a lysis buffer (e.g., Guanidine Hydrochloride) or enzymatic buffer. In some embodiments, the enrichment step can include cell enrichment by both size and selective lysis (e.g., "‘Cell enrichment by size and selective lysis (CESSL)).

[0052] Analyzing a Tarset Cell

[0053] In some embodiments, methods provided herein include analyzing one or more features of a target cell (e.g., an enriched target cell). In some embodiments, one or more features of a target cell (e.g., an enriched target cell) that are analyzed can include a presence of aneuploidy. global hypomethylation, tumor suppressor protein loss, phosphorylation of oncoproteins, elevated proliferation rate, tetraploidy, presence of cancer specific biomarkers (e.g., ALK gene rearrangement, B2M, BRCA1 and BRCA2 gene mutations. CD 19, CD20, CD25, EGFR gene mutation, immunoglobulins. KRAS gene mutation, ROS1 gene rearrangement. T-cell receptor gene rearrangement), and / or elevated nuclear to cytoplasmic ratio. In some embodiments, one or more features of a target cell (e.g., an enriched target cell) that are analyzed can include one or more characteristics of a cancer cell. In some embodiments, a characteristic of a cancer cell includes the presence of aneuploidy in a target cell. In some embodiments, a characteristic of a cancer cell includes global hypomethylation of DNA in a target cell.

[0054] In some embodiments, the analyzing step (c) comprises flow cytometry. In some embodiments, the analyzing step comprises staining a target cell with a fluorescent dye and / or an antibody. In some embodiments, a fluorescent dye can include GelRed. SYBR green, DAPI, Propidium Iodide, or fluorescein. In some embodiments, a fluorescent dye can be conjugated to an antibody. In some embodiments, a fluorescent dye can be conjugated to an antibody, wherein the antibody specifically binds to methylated DNA (e.g., 5 methylcytosine), a tumor suppressor protein (e.g., PTEN, ARID1A, or CDKN2A), or a phosphoryl-group on an oncoprotein (e.g., p-AKT). In some embodiments, a fluorescent dye can be conjugated to an antibody, wherein the antibody specifically binds to a mutant protein (e.g., IDH1 R132H or BRAF V600E) but does not bind to a wild-type protein. In some embodiments, a target cell can be stained with an antibody, wherein the antibody is an anti-CAM 5.2 antibody, an anti- cytokeratin antibody, an anti-CD45 antibody, an anti-CD66b antibody, an estrogen receptor antibody, and / or a progesterone receptor antibody.

[0055] In some embodiments, the analyzing step can comprise genomic sequencing and / or quantitative polymerase chain reaction (qPCR). In some embodiments, the analyzing step can comprise next-generation-sequencing (NGS) techniques, microarrays, and / or parallel real-time PCR. EXAMPLES

[0056] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0057] Materials and Methods

[0058] Tumor cell enrichment

[0059] If cell dissociation was necessary, the sample was placed in 2 rnL of ThinPrep in a Mitlenyi C-top tube and subjected to a custom dissociation protocol on the Miltenyi Octodissociator Machine that involved blending at speeds up to 4000 rpm. The cells were then pelleted by centrifugation and resuspended in 4 mL of phosphate buffered saline containing 1% bovine serum albumin (hereafter, ‘'washing buffer”). If cell dissociation was not deemed necessary', the cells were pelleted directly from the cytology' preservative and resuspended in 4 mL of washing buffer.

[0060] After being resuspend in washing buffer, the cells were passed through a large pore filter, with pore sizes ranging from 15 microns to 30 microns depending on the sample ty pe. These filters may' be obtained from PluriSelect (ex. Pluri Strainer) or Mitlenyi (ex. Preseparation filters) (FIG. 2).

[0061] This step removes superficial squamous epithelial cells. The cells of interest (e.g., urothelial cells, endometrial cells) were then collected on the top of a 5 micron filter, with some proportion of the white blood cells passing through this filter. A lysis buffer was then applied to the top of the 5 micron filter and passed through by generating vacuum pressure under the filter using a syringe. This step removes residual white blood cells.

[0062] The 5 micron filter was then inverted and 4 mL of washing buffer was passed through the filter in reverse to elute the cells of interest into a new tube. The cells were then pelleted by centrifugation from the tube and collected for analysis. Analysis may consist of a sequencing, qPCR, or flow cytometry' method (FIGs. 3-5).

[0063] Flow cytometry analysis

[0064] For flow cytometry' analysis, the cells were pelleted from the cytology7preservative and resuspended in 1 mL PBS / BSA. Chicken Red Blood cells were also added to this solution as a control with known DNA content (FIGs. 11 and 13). If upstream enrichment had not already been performed, this mixture was passed through a 20 micron filter into a collection tube to remove tissue fragments and reduce doublets. The cells were then pelleted by centrifugation. For some protocols (e.g., for urothelial cancer detection), the cells were then fixed in 4% paraformaldehyde for 30 minutes and penneabilized in a solution containing washing buffer with the addition of 0.5% Triton X-100 and 0.5% Tween-20 for 30 minutes. After these successive incubations, the cells were pelleted, washed with washing buffer by centrifugation, and resuspended in a solution containing 2 Normal hy drochloric acid. They were incubated in the acid for 30 minutes at 37 degrees Celsius. The acid was then neutralized by the addition of Tris pH 8.0. The cells were then incubated with the 5mC [33D3] antibody conjugated to the APC fluorophore for 75 minutes at room temperature. After the 75 minute mark, the antibody to CAM5.2 conjugated to the BV421 fluorophore was added. The incubation with both antibodies then continued for 15 minutes. After the incubation, the antibodies were removed by washing the cells by centrifugation with washing buffer (FIGs. 12 and 14).

[0065] The cells were then resuspended in a washing buffer solution containing Propidium Iodide and fluorescein, which stain DNA and total protein, respectively. After incubation with these stains, the sample was then run on a flow cytometer that is equipped with Violet, Blue, and Red lasers. (Table 1, FIGs. 11-14)

[0066] [Table 1]

[0067] In other protocols (e.g., for endometrial cancer or head and neck squamous cell cancer detection), the cells were pelleted from the cytology preservative and resuspended in 1 mL PBS / BSA. Chicken Red Blood cells were also added to this solution as a control with known DNA content. If upstream enrichment had not already been performed, this mixture w as passed through a 30 micron filter into a collection tube to remove tissue fragments and reduce doublets. The cells were then pelleted by centrifugation.

[0068] The cells were then stained with antibodies against the PTEN tumor suppressor protein or other markers, such as the ARID I A tumor suppressor protein or the phospho- AKT protein. After the antibody incubation period, the cells w ere pelleted and washed with w ashing buffer. The cells were then resuspended in a washing buffer solution containing DAPI and fluorescein, which stain DNA and total protein, respectively. After incubation with these stains, the sample was then run on a flow cytometer that is equipped with Violet, Blue, and Red lasers.

[0069] Example 1 - Enrichment and analysis of urothelial tumor cells from urine sample

[0070] A pre-analytic method was performed for enrichment of tumor cells based on their differences in size and susceptibility to lysis compared to the two normal cell types present in a biological fluid sample from a subject (leukocytes and squamous cells) (FIG. 1). Tumor cells shed from cells in tissue fragments were dissociated into single cells so their size properties could be used for enrichment. The tumor cell enrichment and dissociation methods were compatible with clinically used alcohol-based cytology preservatives, including ThinPrep and SurePath preservatives.

[0071] A flow-cytometry based assay was performed for the detection of tumor cells based on the presence of aneuploidy, global hypomethylation, and / or tumor suppressor protein loss. This method works by staining cells with fluorescent dyes specific for DNA, and / or antibodies against methylated DNA, and / or tumor suppressor proteins. The cells were then passed through a flow cytometer and the level of each analyte per cell was measured. Cancer cells can be identified as having abnormal quantities of one or more analytes compared to normal cells. In principle, the method can also be used to detect other cancer specific biomarkers, such as phosphorylated oncoproteins, an elevated proliferation rate, tetraploidy, or an elevated nuclear to cytoplasmic ratio, among others.

[0072] The tumor cells were analyzed to assess cancer-specific changes in DNA, methylated DNA, and / or tumor suppressor protein levels specifically in the subpopulation that is enriched for cells within a sample that give rise to the cancer of interest (such as urothelial cells and endometrial cells in urine and pap test fluid for the detection of urothelial and endometrial cancers, respectively). This works by staining that subpopulation with an antibody, such as CAM 5.2, and / or identifying it based on its physical properties as detected by flow cytometry. Cancer-specific changes in the level of each analyte can then be assessed specifically within that cell population (FIGs. 6-10).

[0073] The flow cytometry assay was compatible with cells stored in the clinically used alcohol-based preservative SurePath. This includes voided urine, pap test samples, and saliva. In principle, it is compatible with any cell-based sample in which tumor cells are admixed with normal cells.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting a cancer in a subject, the method comprising:(a) obtaining a biological sample from the subject, wherein the biological sample comprises a plurality of cells;(b) enriching a target cell from the plurality of cells; and(c) analyzing the target cell and quantifying one or more features of the target cell, thereby detecting the cancer in the subject.

2. The method of claim 1, further comprising dissociating the plurality of cells into single cells before the enrichment step (b).

3. The method of claim 2, wherein the dissociating and enrichment step comprises using an alcohol-based cytology preservative.

4. The method of any one of claims 1-3, wherein the enrichment step (b) comprises separating the target cell based on the target cell size.

5. The method of any one of claims 1-4, wherein the enrichment step (b) comprises enriching the target cell based on the target cell susceptibility to lysis.

6. The method of any one of claims 1-5, wherein the one or more features comprise a presence of aneuploidy, global hypomethylation, tumor suppressor protein loss, presence of cancer specific biomarkers, phosphorylation of oncoproteins, elevated proliferation rate, tetraploidy, elevated nuclear to cytoplasmic ratio, or any combination thereof.

7. The method of claim 6, wherein the one or more features comprise the presence of aneuploidy in the target cell.

8. The method of claim 6, wherein the one or more features comprises global hypomethylation of DNA in the target cell.

9. The method of any one of claims 1-8, wherein the analyzing step (c) comprises flow cytometry.

10. The method of any one of claims 1-9, wherein the analyzing step (c) comprises staining the target cell with a fluorescent dye and / or an antibody.1 1. The method of claim 10, wherein the fluorescent dye comprises GelRed, SYBR green, DAPI, Propidium Iodide, or fluorescein.

12. The method of claim 11. wherein the fluorescent dye is conjugated to the antibody, wherein the antibody specifically binds to methylated DNA, a tumor suppressor protein, or a phosphoryl-group on an oncoprotein.

13. The method of claim 10, wherein the antibody is an anti-CAM 5.2 antibody, an anti- cytokeratin antibody, an anti-CD45 antibody, an anti-CD66b antibody, an estrogen receptor antibody, or a progesterone receptor antibody.

14. The method of any one of claims 1-13, wherein the biological sample comprises saliva, pap test fluid, urine, cerebrospinal fluid, ascites fluid, or pleural fluid from the subject.

15. The method of claim 14, wherein the biological sample comprises urine from the subject.

16. The method of claim 15, wherein the target cell comprises a urothelial cell.

17. The method of claim 14, wherein the biological sample comprises pap test fluid from the subject.

18. The method of claim 17, wherein the target cell comprises an endometrial cell.

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