# US 2014/0193833 A1 · Isolation and detection of cancer cells

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- Number: US 2014/0193833 A1
- Document ID: US20140193833A1
- Status: Published application
- Kind: A1
- Application number: 14/237,747
- Filed: 2012-08-08
- Published: 2014-07-10
- Earliest claimed: 2011-08-08
- Assignees: The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc.
- Inventors: Shiv K. Srivastava; Petrovics Gyorgy; Shyh-Han Tan; Kristen Nickens
- Web page: https://patentgrep.com/patent/US20140193833A1

## CPC

- G01N 33/57555: of the prostate
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/57525: of the liver or pancreas
- G01N 33/57557: {of other specific parts of the body, e.g. brain}
- G01N 33/57575: {involving oncogenic proteins}
- G01N 2333/47: Assays involving proteins of known structure or function as defined in the subgroups
- G01N 2333/99: Isomerases (5.)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided are methods of using polycarbonate filters to isolate and detect cancer cells in a biological fluid, particularly biological fluids, such as urine, that contain very low concentrations of cancer cells. The characterization of the isolated cells for the presence or absence of cancer specific proteins is useful for cancer diagnosis and prognosis.

## Claims

### Claim 1

A method of detecting a cancer cell in a biological fluid suspected of containing the cancer cell, the method comprising:
incubating the biological fluid with a reagent for fixing cells;
passing the biological fluid through a filter, such that if the biological fluid contains the cancer cell, the cancer cell is retained on the filter, wherein the filter comprises a polycarbonate substrate and a plurality of pores;
washing the filter;
incubating the filter with at least one detectable reagent that binds to the cancer cell to form a detectable complex;
washing the filter; and
detecting the detectable complex directly on the filter, wherein detecting the detectable complex indicates the presence of the cancer cell in the biological fluid.

### Claim 2 (depends on 2)

The method of claim 2, wherein the biological fluid is urine.

### Claim 3 (depends on 2)

The method of claim 2, wherein the cancer cell is a prostate cancer cell, a bladder cancer cell, a kidney cancer cell, a ureter cancer cell, or a urethra cancer cell.

### Claim 4 (depends on 1)

The method of claim 1, wherein the reagent for fixing cells comprises one or more alcohols.

### Claim 5 (depends on 4)

The method of claim 4, wherein the one or more alcohols comprise one or more of methyl alcohol, ethyl alcohol, and isopropyl alcohol.

### Claim 6 (depends on 1)

The method of claim 1, wherein each pore in the plurality of pores has a diameter of about 2 μm to 8 μm.

### Claim 7 (depends on 1)

The method of claim 1, wherein the filter comprises a single layer of polycarbonate substrate and the pores pass straight through the filter.

### Claim 8 (depends on 1)

The method of claim 1, wherein the at least one detectable reagent is at least one antibody or at least one nucleic acid probe.

### Claim 9 (depends on 1)

The method of claim 1, wherein the at least one antibody binds to one of the following human proteins: an Ets Related Gene (ERG) protein, an alpha-methylacyl-CoA racemase (AMACR) protein, a prostate specific antigen (PSA) protein, a nucleolin protein, a phosphatase and tensin homolog (PTEN) protein, a phosphorylated Akt protein, a c-Myc protein, a RAF protein, a p63 protein, a prostate specific membrane antigen (PSMA) protein, a NKX3.1 protein, a high molecular weight cytokeratin, a SPARC protein, a SPINK1 protein, a SPOP protein, a BRAF protein, a p53 protein, a NCOA2 protein, a MAOA protein, a CAMK2N1 protein, COL3A1, CLDN8, HOXC6, TMEFF2, NPY, HPGD, BICD1, OR51E1 (PSGR), OR51E2 (PSGR2), NEFH, MSMB, CACNA1D, PLA2G7, MYO6, CRISP3, TWIST1, JAG1, PCGEM1, PMEPA1, LTF, and ETV1 or ETV4.

### Claim 10 (depends on 9)

The method of claim 9, wherein the at least one antibody binds to an ERG epitope formed by amino acids 42-66 of SEQ ID NO:1.

### Claim 11 (depends on 9)

The method of claim 9, wherein the at least one antibody comprises an antibody that binds to a human ERG3 protein, an antibody that binds to a human AMACR protein, and an antibody that binds to a human PSA protein or a human NKX3.1 protein.

### Claim 12 (depends on 1)

The method of claim 1, wherein the at least one nucleic acid probe detects expression of one of the following human genes: the Ets Related Gene (ERG), alpha-methylacyl-CoA racemase (AMACR), prostate specific antigen (PSA), nucleolin gene, phosphatase and tensin homolog (PTEN), Akt, c-Myc, RAF, p63, prostate specific membrane antigen (PSMA), NKX3.1, a human gene encoding a high molecular weight cytokeratin, SPARC, SPINK1, SPOP, BRAF, p53, NCOA2, MAOA, CAMK2N1, COL3A1, CLDN8, HOXC6, TMEFF2, NPY, HPGD, BICD1, OR51E1 (PSGR), OR51E2 (PSGR2), NEFH, MSMB, CACNA1D, PLA2G7, MYO6, CRISP3, TWIST1, JAG1, PCGEM1, PMEPA1, LTF, and ETV1 or ETV4.

### Claim 13 (depends on 1)

The method of claim 1, wherein as few as 10 cancer cells can be detected in the biological fluid.

### Claim 14 (depends on 1)

The method of claim 1, wherein the method is capable of detecting at least about 70% of cancer cells in a sample containing 100 cancer cells.

### Claim 15 (depends on 1)

The method of claim 1, wherein the method does not include a centrifugation step.

### Claim 16

A method of isolating a cancer cell, the method comprising:
incubating a biological fluid containing the cancer cell with a reagent for fixing cells; and
isolating the cancer cell on a filter by passing the biological fluid through the filter, wherein the filter comprises a polycarbonate substrate and a plurality of pores.

### Claim 17 (depends on 16)

The method of claim 16, wherein the biological fluid is urine.

### Claim 18 (depends on 17)

The method of claim 17, wherein the cancer cell is a prostate cancer cell, a bladder cancer cell, a kidney cancer cell, a ureter cancer cell, or a urethra cancer cell.

### Claim 19 (depends on 16)

The method of claim 16, wherein the reagent for fixing cells comprises one or more alcohols.

### Claim 20 (depends on 19)

The method of claim 19, wherein the one or more alcohols comprise one or more of methyl alcohol, ethyl alcohol, and isopropyl alcohol.

### Claim 21 (depends on 16)

The method of claim 16, wherein each pore in the plurality of pores has a diameter of about 2 μm to 8 μm.

### Claim 22 (depends on 16)

The method of claim 16, wherein the filter comprises a single layer of polycarbonate substrate and the pores pass straight through the filter.

### Claim 23 (depends on 16)

A filter comprising a cancer cell, wherein the filter is obtained through the method of claim 16.