Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7023—(Hyper)proliferation
  • G01N2800/7028—Cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5026—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell morphology
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

• the present disclosure relates generally to methods for selecting a therapy for a metastatic castration resistant prostate cancer (mCRPC) patient comprising detection of Androgen Receptor Variant 7 (AR-V7) in circulating tumor cells (CTCs).
• mCRPC metastatic castration resistant prostate cancer
• AR-V7 Androgen Receptor Variant 7
• PC Prostate cancer
• Circulating tumor cells represent a significant advance in cancer diagnosis made even more attractive by their non-invasive measurement.
• Quantifying and characterizing CTCs, as a liquid biopsy, assists clinicians to select the course of therapy and to watch monitor how a patient's cancer evolves.
• CTCs can therefore be considered not only as surrogate biomarkers for metastatic disease but also as a promising key tool to track tumor changes, treatment response, cancer recurrence or patient outcome non-invasively.
• a key limitation to the predictive value of an AR-V7 mRNA assay in CTCs is the analytical validation of the robustness of low frequency and labile mRNA measurement in CTCs, which may not meet diagnostic workflows amenable to community practices.
• EpCAM- CTCs may lead to undersampling the AR-V7 biomarker.
• the present invention addresses this need and provides related advantages.
• the present disclosure describes an AR-V7 immunofluorescent test for use in fixed single CTCs, inclusive of all CTC subtypes utilizing samples from progressive mCRPC patients in need of a change in therapy.
• the present invention is directed to a method of identifying an mCRPC patient with an improved response to taxane therapy compared to androgen receptor (AR) targeted therapy comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to generate circulating tumor cell (CTC) data, wherein the analysis comprises detecting the presence of an Androgen Receptor Variant 7 (AR-V7) in said cells, and (c) evaluating the CTC data to identify a mCRPC patient with an improved response to taxane therapy compared to Androgen receptor signaling-directed (ARS-directed) therapy.
• ARS-directed Androgen receptor signaling-directed
• the present invention is directed to a method of identifying an mCRPC patient with an improved response to taxane therapy compared to androgen receptor (AR) targeted therapy comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to generate circulating tumor cell (CTC) data, wherein the analysis comprises detecting the presence of an Androgen Receptor Variant 7 (AR-V7) in said cells, and (c) evaluating the CTC data to identify a mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy, and identifying said mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy based on nuclear localization of the AR-V7 in CTCs.
• AR Androgen Receptor Variant 7
• the present invention is directed to a method of identifying an mCRPC patient with an improved response to taxane therapy compared to androgen receptor (AR) targeted therapy comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to generate CTC data, wherein the analysis comprises detecting the presence of an AR-V7 in said cells, and (c) evaluating the CTC data to identify a mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy, and identifying said mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy based on nuclear localization of the AR-V7 in CTCs, wherein the nuclear localization of the AR-V7 corresponds to resistance to ARS-directed therapy.
• AR androgen receptor
• the present invention is directed to a method of identifying an mCRPC patient with an improved response to taxane therapy compared to androgen receptor (AR) targeted therapy comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to generate CTC data, wherein the analysis comprises detecting the presence of an AR-V7 in said cells, and (c) evaluating the CTC data to identify a mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy, and identifying said mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy based on nuclear localization of the AR-V7 in CTCs, wherein the nuclear localization of the AR-V7 corresponds to a positive response to taxane therapy compared to ARS-directed therapy.
• AR androgen receptor
• the nuclear localization comprises a staining pattern with signal intensity >3-fold higher than background staining from neighboring white blood cells (WBCs).
• WBCs white blood cells
• the methods comprise an additional step (d) wherein a patient is treated with taxane therapay if identified as having an improved response to taxane therapy compared to ARS-directed therapy.
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a metastatic castration resistant prostate cancer (mCRPC) patient to generate circulating tumor cell (CTC) data, wherein the analysis comprises detecting the presence of an Androgen Receptor Variant 7 (AR-V7) in said cells.
• mCRPC metastatic castration resistant prostate cancer
• CTC circulating tumor cell
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a mCRPC patient to generate CTC data, wherein the analysis comprises detecting the presence of AR-V7 localized in the nucleus of CTCs.
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a mCRPC patient to generate CTC data, wherein the analysis comprises detecting the presence of AR-V7 localized in the nucleus of CTCs, wherein the nuclear localization of the AR-V7 corresponds to resistance to ARS-directed therapy for the mCRPC patient.
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a mCRPC patient to generate CTC data, wherein the analysis comprises detecting the presence of AR-V7 localized in the nucleus of CTCs, wherein the nuclear localization of the AR-V7 corresponds to a positive response to taxane therapy compared to ARS-directed therapy for the mCRPC patient.
• the nuclear localization comprises a staining pattern with signal intensity >3-fold higher than background staining from neighboring white blood cells (WBCs).
• WBCs white blood cells
• FIG. 1 A shows post-therapy PSA change patterns after AR signaling directed therapies.
• 193 mCRPC patient blood samples were collected prior to starting Abiraterone (44); Enzalutamide (81), Docetaxel (46) Cabazitaxel (13), and Paclitaxel (2).
• PSA Outcomes were recorded as Sensitive (S): Patterns 1 and 2, or Resistant (R): Pattern 3 (A).
• S Sensitive
• R Resistant
• Patients were monitored for up to 2.3 yrs to assess rPFS and OS outcomes.
• Figures 2A and 2B show AR-V7 IF assay development.
• a rabbit monoclonal antibody specific to the AR-V7 splice variant was used for IF staining of cell line controls and patient CTCs characterized by the Epic CTC Platform. Positive (22RV1), Figure 2A, upper, and negative (DU145), Figure 2A, lower, cell line controls were used to assess initial AR-V7 IF protein assay.
• AR-V7 positive cells were defined as cells having a specific nuclear-localized staining pattern with signal intensity >3-fold higher than background staining from neighboring WBCs.
• Figure 2B depicts a graph showing that 55% (2660/4813) of individually characterized 22RV1 cells showed specific AR-V7 staining, while nuclear staining was seen in only 0.001% (5/3360) of DU145 negative controls.
• Figures 3 A and 3B show patient demographics.
• Figure 3 A shows patient characteristics
• Figure 3B shows patient line of therapy at the time the samples were obtained.
• Figures 4A-4C show that baseline AR-V7+ CTCs predict PSA resistance to AR therapy but not to taxane chemotherapy.
• Figure 4A shows representative CTC images from a single 3+ Line patient demonstrating AR-V7 and CTC heterogeneity.
• Figure 4B shows graphs depicting the number of AR-V7 positive CTCs per ml in AR therapy resistant and sensitive patients (left) and the number of AR-V7 positive CTCs per ml in taxane therapy resistant and sensitive patients (right).
• Figure 4C shows a table demonstrating that AR-V7 positivity predicts PSA resistance to AR-directed therapy. Briefly, AR-V7 protein expression was found in CTCs from 15/66 patients resistant to AR Therapy.
• Figures 5A-5C demonstrate that baseline AR-V7+ CTCs predict unfavorable outcomes on AR therapy and that, in patients on AR-directed therapies, AR-V7 positive status is associated with shorter time on therapy ( Figure 5 A), shorter radiographic
• Figures 6A-6D show heterogeneity and prevalence of AR-V7+ CTCs, and specifically shows that frequency and heterogeneity of AR-V7 positivity increased in patients with increasing lines of therapy.
• CTCs were identified in 144/191 (75%) patients.
• AR-V7+ CTCs were found in 2/67 (3%) first line (Figure 6A) , 9/50 (18%) second line (Figure 6B), and 23/74 (31%) third or greater lines (Figure 6C).
• Figure 6D depicts a table showing that frequency and heterogeneity of AR-V7 positivity increased in patients with increasing lines of therapy. DETAILED DESCRIPTION
• the present disclosure describes an AR-V7 immunofluorescent test for use in fixed single CTCs, inclusive of all CTC subtypes utilizing samples from progressive mCRPC patients in need of a change in therapy on a platform designed for global diagnostic workflows.
• the present invention provides a method of identifying a metastatic castration resistant prostate cancer (mCRPC) patient with an improved response to taxane therapy compared to androgen receptor (AR) targeted therapy comprising (a) performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from the patient to generate circulating tumor cell (CTC) data, wherein the analysis comprises detecting the presence of an Androgen Receptor Variant 7 (AR-V7) in said cells, and (c) evaluating the CTC data to identify a mCRPC patient with an improved response to taxane therapy compared to ARS-directed therapy therapy.
• mCRPC metastatic castration resistant prostate cancer
• AR androgen receptor
• CTC circulating tumor cell
• CTCs which can be present as single cells or in clusters of CTCs, are often epithelial cells shed from solid tumors found in very low concentrations in the circulation of patients.
• a traditional CTC refers to a single CTC that is cytokeratin positive, CD45 negative, contains a DAPI nucleus, and is morphologically distinct from surrounding white blood cells.
• a non-traditional CTC refers to a CTC that differs from a traditional CTC in at least one characteristic.
• Non-traditional CTCs include the five CTC subtypes shown in Figure 2, Panel B, including CTC clusters, CK negative CTCs that are positive at least one additional biomarker that allows classification as a CTC , small CTCs, nucleoli + CTCs and CK speckled CTCs.
• CTC cluster means two or more CTCs with touching cell membranes.
• a biological sample can be any sample that contains CTCs.
• a sample can comprise a bodily fluid such as blood; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint; cells; skin, and the like.
• a biological sample obtained from a subject can be any sample that contains cells and encompasses any material in which CTCs can be detected.
• a sample can be, for example, whole blood, plasma, saliva or other bodily fluid or tissue that contains cells.
• the biological sample is a blood sample.
• a sample can be whole blood, more preferably peripheral blood or a peripheral blood cell fraction.
• a blood sample can include any fraction or component of blood, without limitation, T-cells, monocytes, neutrophiles, erythrocytes, platelets and microvesicles such as exosomes and exosome-like vesicles.
• blood cells included in a blood sample encompass any nucleated cells and are not limited to components of whole blood.
• blood cells include, for example, both white blood cells (WBCs) as well as rare cells, including CTCs.
• WBCs white blood cells
• the samples of this disclosure can each contain a plurality of cell populations and cell subpopulation that are distinguishable by methods well known in the art (e.g., FACS, immunohistochemistry).
• a blood sample can contain populations of non- nucleated cells, such as erythrocytes (e.g., 4-5 million/ ⁇ ) or platelets (150,000-400,000 cells/ ⁇ ), and populations of nucleated cells such as WBCs (e.g., 4,500 - 10,000 cells/ ⁇ ), CECs or CTCs (circulating tumor cells; e.g., 2-800 cells/).
• WBCs may contain cellular subpopulations of, e.g., neutrophils (2,500-8,000 cells/ ⁇ ), lymphocytes (1,000-4,000 cells/ ⁇ ), monocytes (100-700 cells/ ⁇ ), eosinophils (50-500 cells/ ⁇ ), basophils (25 - 100 cells/ ⁇ ) and the like.
• the samples of this disclosure are non-enriched samples, i.e., they are not enriched for any specific population or subpopulation of nucleated cells.
• non-enriched blood samples are not enriched for CTCs, WBC, B-cells, T-cells, K-cells, monocytes, or the like.
• the methods can be performed by methods known to those skilled in the art, for example, as described by Marrinucci et al. Hum Pathol 38(3): 514-519 (2007); Marrinucci et al. Arch Pathol Lab Med 133(9): 1468-1471 (2009); Mikolajczyk et al. J Oncol 2011 :
• the term "direct analysis” means that the CTCs are detected in the context of all surrounding nucleated cells present in the sample as opposed to after enrichment of the sample for CTCs prior to detection.
• the methods comprise microscopy providing a field of view that includes both CTCs and at least 200 surrounding white blood cells (WBCs).
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a metastatic castration resistant prostate cancer (mCRPC) patient to generate circulating tumor cell (CTC) data, wherein the analysis comprises detecting the presence of an Androgen Receptor Variant 7 (AR-V7) in said cells.
• mCRPC metastatic castration resistant prostate cancer
• CTC circulating tumor cell
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a mCRPC patient to generate CTC data, wherein the analysis comprises detecting the presence of AR-V7 localized in the nucleus of CTCs.
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a mCRPC patient to generate CTC data, wherein the analysis comprises detecting the presence of AR-V7 localized in the nucleus of CTCs, wherein the nuclear localization of the AR-V7 corresponds to resistance to ARS-directed therapy for the mCRPC patient.
• the present invention also provides a method of performing a direct analysis comprising immunofluorescent staining and morphological characterization of nucleated cells in a blood sample obtained from a mCRPC patient to generate CTC data, wherein the analysis comprises detecting the presence of AR-V7 localized in the nucleus of CTCs, wherein the nuclear localization of the AR-V7 corresponds to a positive response to taxane therapy compared to ARS-directed therapy for the mCRPC patient.
• the nuclear localization of AR-V7 comprises a staining pattern with signal intensity about >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, >10-fold, >11-fold, >12-fold, >13-fold, >14-fold, >15-fold, >20-fold, >25-fold, >30-fold, or >35-fold higher than background staining from neighboring white blood cells (WBCs).
• WBCs white blood cells
• a fundamental aspect of the present disclosure is the unparalleled robustness of the disclosed methods with regard to the detection of CTCs.
• the rare event detection disclosed herein with regard to CTCs is based on a direct analysis, i.e. non-enriched, of a population that encompasses the identification of rare events in the context of the surrounding non-rare events. Identification of the rare events according to the disclosed methods inherently identifies the surrounding events as non-rare events. Taking into account the surrounding non-rare events and determining the averages for non-rare events, for example, average cell size of non-rare events, allows for calibration of the detection method by removing noise.
• the result is a robustness of the disclosed methods that cannot be achieved with methods that are not based on direct analysis, but that instead compare enriched populations with inherently distorted contextual comparisons of rare events.
• the robustness of the direct analysis methods disclosed herein enables characterization of CTC, including subtypes of CTCs described herein, that allows for identification of phenotypes and heterogeneity that cannot be achieved with other CTC detection methods and that enables the analysis of biomarkers in the context of the claimed methods.
• CTC data can include both morphological features and immunofluorescent features.
• biomarkers can include a biological molecule, or a fragment of a biological molecule, the change and/or the detection of which can be correlated, individually or combined with other measurable features, with prostate cancer and/or mCRPC.
• CTCs which can be present a single cells or in clusters of CTCs, are often epithelial cells shed from solid tumors and are present in very low
• CTCs have an abundance of less than 1 : 1,000 in a blood cell population, e.g., an abundance of less than 1 :5,000, 1 : 10,000,
• the a CTC has an abundance of 1 :50:000 to 1 : 100,000 in the cell population.
• the samples of this disclosure may be obtained by any means, including, e.g., by means of solid tissue biopsy or fluid biopsy ⁇ see, e.g., Marrinucci D. et al, 2012, Phys. Biol. 9 016003). Briefly, in particular embodiments, the process can encompass lysis and removal of the red blood cells in a 7.5 mL blood sample, deposition of the remaining nucleated cells on specialized microscope slides, each of which accommodates the equivalent of roughly 0.5 mL of whole blood.
• a blood sample may be extracted from any source known to include blood cells or components thereof, such as venous, arterial, peripheral, tissue, cord, and the like.
• the samples may be processed using well known and routine clinical methods (e.g., procedures for drawing and processing whole blood).
• a blood sample is drawn into anti-coagulent blood collection tubes (BCT), which may contain EDTA or Streck Cell-Free DNATM.
• BCT anti-coagulent blood collection tubes
• a blood sample is drawn into CellSave® tubes (Veridex).
• a blood sample may further be stored for up to 12 hours, 24 hours, 36 hours, 48 hours, or 60 hours before further processing.
• the methods of this disclosure comprise an intitial step of obtaining a white blood cell (WBC) count for the blood sample.
• WBC white blood cell
• the WBC count may be obtained by using a HemoCue® WBC device (Hemocue, Angelholm, Sweden).
• the WBC count is used to determine the amount of blood required to plate a consistent loading volume of nucleated cells per slide and to calculate back the equivalent of CTCs per blood volume.
• the methods of this disclosure comprise an initial step of lysing erythrocytes in the blood sample.
• the erythrocytes are lysed, e.g., by adding an ammonium chloride solution to the blood sample.
• a blood sample is subjected to centrifugation following erythrocyte lysis and nucleated cells are resuspended, e.g., in a PBS solution.
• nucleated cells from a sample are deposited as a monolayer on a planar support.
• the planar support may be of any material, e.g., any fluorescently clear material, any material conducive to cell attachment, any material conducive to the easy removal of cell debris, any material having a thickness of ⁇ 100 ⁇ .
• the material is a film.
• the material is a glass slide.
• the method encompasses an initial step of depositing nucleated cells from the blood sample as a monolayer on a glass slide.
• the glass slide can be coated to allow maximal retention of live cells (See, e.g., Marrinucci D.
• the methods of this disclosure comprise depositing about 3 million cells onto a glass slide. In additional embodiments, the methods of this disclosure comprise depositing between about 2 million and about 3 million cells onto the glass slide. In some embodiments, the glass slide and immobilized cellular samples are available for further processing or experimentation after the methods of this disclosure have been completed. [0045] In some embodiments, the methods of this disclosure comprise an initial step of identifying nucleated cells in the non-enriched blood sample. In some embodiments, the nucleated cells are identified with a fluorescent stain. In certain embodiments, the
• fluorescent stain comprises a nucleic acid specific stain.
• the fluorescent stain is diamidino-2-phenylindole (DAPI).
• DAPI diamidino-2-phenylindole
• immunofluorescent staining of nucleated cells comprises pan cytokeratin (CK), cluster of differentiation (CD) 45 and DAPI.
• CTCs comprise distinct immunofluorescent staining from surrounding nucleated cells.
• the distinct immunofluorescent staining of CTCs comprises DAPI (+), CK (+) and CD 45 (-).
• the identification of CTCs further comprises comparing the intensity of pan cytokeratin fluorescent staining to surrounding nucleated cells.
• the CTC data is generated by fluorescent scanning microscopy to detect immunofluorescent staining of nucleated cells in a blood sample. Marrinucci D. et al, 2012, Phys. Biol. 9 016003).
• CK cytokeratin
• DAPI nuclear stain
• the nucleated blood cells can be imaged in multiple fluorescent channels to produce high quality and high resolution digital images that retain fine cytologic details of nuclear contour and cytoplasmic distribution.
• CTCs can be identified as DAPI (+), CK (+) and CD 45 (-).
• the CTCs comprise distinct immunofluorescent staining from surrounding nucleated cells.
• the CTC data includes traditional CTCs also known as high definition CTCs (HD-CTCs) .
• Traditional CTCs are CK positive, CD45 negative, contain an intact DAPI positive nucleus without identifiable apoptotic changes or a disrupted appearance, and are morphologically distinct from surrounding white blood cells (WBCs).
• WBCs white blood cells
• DAPI (+), CK (+) and CD45 (-) intensities can be categorized as measurable features during HD-CTC enumeration as previously described. Nieva et al., Phys Biol 9:016004 (2012).
• the enrichment-free, direct analysis employed by the methods disclosed herein results in high sensitivity and high specificity, while adding high definition cytomorphology to enable detailed morphologic characterization of a CTC population known to be heterogeneous.
• CTCs can be identified as comprises DAPI (+), CK (+) and CD 45 (-) cells
• the methods of the invention can be practiced with any other biomarkers that one of skill in the art selects for generating CTC data and/or identifying CTCs and CTC clusters.
• One skilled in the art knows how to select a morphological feature, biological molecule, or a fragment of a biological molecule, the change and/or the detection of which can be correlated with a CTC.
• Molecule biomarkers include, but are not limited to, biological molecules comprising nucleotides, nucleic acids, nucleosides, amino acids, sugars, fatty acids, steroids, metabolites, peptides, polypeptides, proteins, carbohydrates, lipids, hormones, antibodies, regions of interest that serve as surrogates for biological macromolecules and combinations thereof (e.g., glycoproteins, ribonucleoproteins, lipoproteins).
• the term also encompasses portions or fragments of a biological molecule, for example, peptide fragment of a protein or polypeptide
• CTC data including microscopy based approaches, including fluorescence scanning microscopy ⁇ see, e.g., Marrinucci D. et al, 2012, Phys. Biol. 9 016003), mass spectrometry approaches, such as MS/MS, LC-MS/MS, multiple reaction monitoring (MRM) or SRM and product-ion monitoring (PIM) and also including antibody based methods such as
• Immunoassay techniques and protocols are generally known to those skilled in the art ( Price and Newman, Principles and Practice of Immunoassay, 2nd Edition, Grove's Dictionaries, 1997; and Gosling, Immunoassays: A Practical Approach, Oxford University Press, 2000.)
• a variety of immunoassay techniques, including competitive and noncompetitive immunoassays, can be used (Self et al, Curr. Opin. Biotechnol., 7:60-65 (1996), see also John R.
• biomarkers may be detected using any class of marker-specific binding reagents known in the art, including, e.g., antibodies, aptamers, fusion proteins, such as fusion proteins including protein receptor or protein ligand components, or biomarker-specific small molecule binders.
• marker-specific binding reagents known in the art, including, e.g., antibodies, aptamers, fusion proteins, such as fusion proteins including protein receptor or protein ligand components, or biomarker-specific small molecule binders.
• the presence or absence of CK, AR-V7 or CD45 is determined by an antibody.
• the antibodies of this disclosure bind specifically to a biomarker.
• the antibody can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986).
• the antibody can be any immunoglobulin or derivative thereof, whether natural or wholly or partially
• the antibody has a binding domain that is homologous or largely homologous to an immunoglobulin binding domain and can be derived from natural sources, or partly or wholly synthetically produced.
• the antibody can be a monoclonal or polyclonal antibody.
• an antibody is a single chain antibody.
• antibody can be provided in any of a variety of forms including, for example, humanized, partially humanized, chimeric, chimeric humanized, etc.
• the antibody can be an antibody fragment including, but not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
• the antibody can be produced by any means.
• the antibody can be enzymatically or chemically produced by fragmentation of an intact antibody and/or it can be recombinantly produced from a gene encoding the partial antibody sequence.
• the antibody can comprise a single chain antibody fragment.
• the antibody can comprise multiple chains which are linked together, for example, by disulfide linkages, and any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule. Because of their smaller size as functional components of the whole molecule, antibody fragments can offer advantages over intact antibodies for use in certain
• a detectable label can be used in the methods described herein for direct or indirect detection of the biomarkers when generating CTC data in the methods of the invention.
• a wide variety of detectable labels can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Those skilled in the art are familiar with selection of a suitable detectable label based on the assay detection of the biomarkers in the methods of the invention.
• Suitable detectable labels include, but are not limited to, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, Alexa Fluor® 647, Alexa Fluor® 555, Alexa Fluor® 488), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, and the like.
• fluorescent dyes e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC),
• differential tagging with isotopic reagents e.g., isotope-coded affinity tags (ICAT) or the more recent variation that uses isobaric tagging reagents, iTRAQ (Applied Biosystems, Foster City, Calif), followed by multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis can provide a further methodology in practicing the methods of this disclosure.
• ICAT isotope-coded affinity tags
• iTRAQ Applied Biosystems, Foster City, Calif
• MS/MS tandem mass spectrometry
• a chemiluminescence assay using a chemiluminescent antibody can be used for sensitive, non-radioactive detection of proteins.
• An antibody labeled with fluorochrome also can be suitable.
• fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine.
• Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase, urease, and the like. Detection systems using suitable substrates for horseradish-peroxidase, alkaline phosphatase, beta.-galactosidase are well known in the art.
• a signal from the direct or indirect label can be analyzed, for example, using a microscope, such as a fluorescence microscope or a fluorescence scanning microscope.
• a spectrophotometer can be used to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125 I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
• assays used to practice the methods of this disclosure can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
• the biomarkers are immunofluorescent markers.
• the immunofluorescent makers comprise a marker specific for epithelial cells
• the immunofluorescent makers comprise a marker specific for white blood cells (WBCs).
• WBCs white blood cells
• one or more of the immunofluorescent markers comprise CD 45 and CK.
• the presence or absence of immunofluorescent markers in nucleated cells results in distinct immunofluorescent staining patterns.
• Immunofluorescent staining patterns for CTCs and WBCs may differ based on which epithelial or WBC markers are detected in the respective cells.
• determining presence or absence of one or more immunofluorescent markers comprises comparing the distinct immunofluorescent staining of CTCs with the distinct immunofluorescent staining of CTCs with the distinct
• CTCs comprise distinct morphological characteristics compared to surrounding nucleated cells.
• the morphological characteristics comprise nucleus size, nucleus shape, cell size, cell shape, and/or nuclear to cytoplasmic ratio.
• the method further comprises analyzing the nucleated cells by nuclear detail, nuclear contour, presence or absence of nucleoli, quality of cytoplasm, quantity of cytoplasm, intensity of immunofluorescent staining patterns.
• the morphological characteristics of this disclosure may include any feature, property, characteristic, or aspect of a cell that can be determined and correlated with the detection of a CTC.
• CTC data can be generated with any microscopic method known in the art.
• the method is performed by fluorescent scanning microscopy.
• the microscopic method provides high-resolution images of CTCs and their surrounding WBCs ⁇ see, e.g., Marrinucci D. et al, 2012, Phys. Biol. 9 016003)).
• a slide coated with a monolayer of nucleated cells from a sample is scanned by a fluorescent scanning microscope and the fluorescence intensities from immunofluorescent markers and nuclear stains are recorded to allow for the determination of the presence or absence of each immunofluorescent marker and the assessment of the morphology of the nucleated cells.
• a CTC data includes detecting one or more biomarkers, for example, CK, AR-V7 and CD 45.
• a biomarker is considered "present" in a cell if it is detectable above the background noise of the respective detection method used (e.g., 2-fold, 3-fold, 5-fold, or 10-fold higher than the background; e.g., 2 ⁇ or 3 ⁇ over background).
• a biomarker is considered "absent” if it is not detectable above the background noise of the detection method used (e.g., ⁇ 1.5-fold or ⁇ 2.0-fold higher than the background signal; e.g., ⁇ 1.5 ⁇ or ⁇ 2.0 ⁇ over background).
• the presence or absence of immunofluorescent markers in nucleated cells is determined by selecting the exposure times during the fluorescence scanning process such that all immunofluorescent markers achieve a pre-set level of fluorescence on the WBCs in the field of view. Under these conditions, CTC-specific immunofluorescent markers, even though absent on WBCs are visible in the WBCs as background signals with fixed heights. Moreover, WBC-specific immunofluorescent markers that are absent on CTCs are visible in the CTCs as background signals with fixed heights.
• a cell is considered positive for an immunofluorescent marker (i.e., the marker is considered present) if its fluorescent signal for the respective marker is significantly higher than the fixed background signal (e.g., 2-fold, 3-fold, 5-fold, or 10-fold higher than the background; e.g., 2 ⁇ or 3 ⁇ over background).
• a nucleated cell is considered CD 45 positive (CD 45 + ) if its fluorescent signal for CD 45 is significantly higher than the background signal.
• a cell is considered negative for an immunofluorescent marker (i.e., the marker is considered absent) if the cell's fluorescence signal for the respective marker is not significantly above the background signal (e.g., ⁇ 1.5-fold or ⁇ 2.0-fold higher than the background signal; e.g., ⁇ 1.5 ⁇ or ⁇ 2.0 ⁇ over background).
• each microscopic field contains both CTCs and WBCs.
• the microscopic field shows at least 1, 5, 10, 20, 50, or 100 CTCs.
• the microscopic field shows at least 10, 25, 50, 100, 250, 500, or 1,000 fold more WBCs than CTCs.
• the microscopic field comprises one or more CTCs or CTC clusters surrounded by at least 10, 50, 100, 150, 200, 250, 500, 1,000 or more WBCs.
• generation of the CTC data comprises enumeration of CTCs that are present in the blood sample.
• the methods described herein encompass detection of at least 1.0 CTC/mL of blood, 1.5 CTCs/mL of blood, 2.0 CTCs/mL of blood, 2.5 CTCs/mL of blood, 3.0 CTCs/mL of blood, 3.5 CTCs/mL of blood, 4.0 CTCs/mL of blood, 4.5 CTCs/mL of blood, 5.0
• CTCs/mL of blood 5.5 CTCs/mL of blood, 6.0 CTCs/mL of blood, 6.5 CTCs/mL of blood, 7.0 CTCs/mL of blood, 7.5 CTCs/mL of blood, 8.0 CTCs/mL of blood, 8.5 CTCs/mL of blood, 9.0 CTCs/mL of blood, 9.5 CTCs/mL of blood, 10 CTCs/mL of blood, or more.
• generation of the CTC data comprises detecting distinct subtypes of CTCs, including non-traditional CTCs.
• the methods described herein encompass detection of at least 0.1 CTC cluster/mL of blood, 0.2 CTC clusters/mL of blood, 0.3 CTC clusters/mL of blood, 0.4 CTC clusters/mL of blood, 0.5 CTC clusters/mL of blood, 0.6 CTC clusters/mL of blood, 0.7 CTC clusters/mL of blood, 0.8 CTC clusters/mL of blood, 0.9 CTC clusters/mL of blood, 1 CTC cluster/mL of blood, 2 CTC clusters/mL of blood, 3 CTC clusters/mL of blood, 4 CTC clusters/mL of blood, 5 CTC clusters/mL of blood, 6 CTC clusters/mL of blood, 7 CTC clusters/mL of blood, 8 CTC clusters/mL of blood, 9 CTC clusters/
• the methods comprise genomic analysis of the CTCs, for example, by fluorescence in situ hybridization (FISH).
• FISH fluorescence in situ hybridization
• PCR Polymerase chain reaction
• Any method capable of determining a DNA copy number profile of a particular sample can be used for molecular profiling according to the invention provided the resolution is sufficient to identify the biomarkers of the invention.
• the skilled artisan is aware of and capable of using a number of different platforms for assessing whole genome copy number changes at a resolution sufficient to identify the copy number of the one or more biomarkers of the invention.
• In situ hybridization assays are well known and are generally described in Angerer et al., Methods Enzymol. 152:649-660 (1987).
• cells e.g., from a biopsy, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled.
• the probes are preferably labeled with radioisotopes or fluorescent reporters.
• fluorescent in situ hybridization uses fluorescent probes that bind to only those parts of a sequence with which they show a high degree of sequence similarity.
• FISH is a cytogenetic technique used to detect and localize specific
• FISH can be used to detect DNA sequences on chromosomes. FISH can also be used to detect and localize specific RNAs, e.g., mRNAs, within tissue samples. In FISH uses fluorescent probes that bind to specific nucleotide sequences to which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out whether and where the fluorescent probes are bound. In addition to detecting specific nucleotide sequences, e.g., translocations, fusion, breaks, duplications and other chromosomal abnormalities, FISH can help define the spatial-temporal patterns of specific gene copy number and/or gene expression within cells and tissues.
• Example 1 Presence of AR-V7 splice variant in the CTCs of mCRPC patients identifies patients with improved PSA response with taxane therapy over androgen receptor signaling directed therapies (ARS Tx)
• ARS Tx taxane therapy over androgen receptor signaling directed therapies
• Circulating tumour cells were identified by immunofluorescence, as described (Marrinucci et al, 2007, supra; Marrinucci et al, 2009, supra; Mikolajczyk et al, 2011, supra; Marrinucci et al, 2012, supra; Werner et al, 2015, supra).
• AR-V7 protein expression was found in CTCs from 15/66 patients resistant to AR Therapy. While CTCs were identified in 37/57 AR Therapy sensitive patients (range: is 0 to 345 CTCs/mL , median: 2 CTCs/mL), 0/57 harbored AR-V7+ CTCs. Of all patients in the AR Therapy cohort that lacked AR-V7+ cells, 53% were sensitive to AR Therapy.
• AR-V7 prevalence does not predict resistance to Taxane chemotherapy: AR-V7+ CTCs were found in 9/30 and 7/26 Taxane-sensitive and resistant patients, respectively. Figures 4 and 5.
• AR-V7 expression is not associated with PSA resistance in taxane treated patients.
• AR-V7 prevalence increases with increased exposure to systemic therapy AR-V7 (p ⁇ 0.0001) and represents a minority population of total CTCs suggestive of disease heterogeneity.
• Figure 6 shows that AR-V7 prevalence increases with increased exposure to systemic therapy AR-V7 (p ⁇ 0.0001) and represents a minority population of total CTCs suggestive of disease heterogeneity.
• This example demonstrates the association between the presence of AR-V7 positive CTCs and AR therapy resistance but not taxane resistance, confirming the utilization of the AR-V7 biomarker to inform treatment selection in mCRPC patients.

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