# US 11,898,209 B2 · Digital analysis of circulating tumor cells in blood samples

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- Number: US 11,898,209 B2
- Document ID: US11898209B2
- Status: Granted
- Kind: B2
- Application number: 17/065,889
- Filed: 2020-10-08
- Published: 2021-06-24
- Granted: 2024-02-13
- Earliest claimed: 2015-03-25
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: Daniel A. Haber; Ravi Kapur; Mehmet Toner; Shyamala Maheswaran; Xin Hong; David Tomoaki Miyamoto; Tanya Todorova; Sarah Javaid
- Web page: https://patentgrep.com/patent/US11898209B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

This disclosure relates to new assay methods for analysis of circulating tumor cells (CTCs) in blood samples for detection, e.g., early detection, and/or monitoring of disease, e.g., cancer. The methods provide ultra-high sensitivity and specificity, and include the use of microfluidic isolation of CTCs and digital detection of RNA derived from the CTCs.

## Claims

### Claim 1

A method for analyzing circulating tumor cells (CTCs) in a blood sample from a subject, the method comprising:
isolating circulating tumor cells (CTCs) from the blood sample;
isolating ribonucleic acid (RNA) from the CTC;
generating cDNA molecules in solution from the isolated RNA;
encapsulating cDNA molecules into individual droplets;
amplifying cDNA within each of the individual droplets in the presence of one or more reporter groups configured to bind specifically to cDNA corresponding to tumor lineage-specific RNA from a specific type of tissue that is the source of the CTCs and not to bind to cDNA from normal cells in the blood;
detecting droplets that contain the reporter groups as an indicator of the presence of amplified cDNA molecules from CTCs in the droplets; and
analyzing cDNA molecules from CTCs in the detected droplet;
wherein amplifying cDNA molecules within each of the individual droplets comprises conducting PCR in each droplet,
wherein at least one primer set for each type of cancer is used for amplifying the cDNA molecules within each of the droplets, wherein each primer set corresponds to a selected cancer gene,
wherein the selected cancer genes include prostate cancer-selective genes or breast cancer-selective genes,
wherein the prostate cancer-selective genes are FAT1, TMPRSS2, AGR2, FOLH1, HOXDB13, KLK2, KLK3, and STEAP2,
wherein the primer sets corresponding to the prostate cancer-selective genes are:
FAT1 (primer 1 SEQ ID NO:23, primer 2 SEQ ID NO:22),
TMPRSS2 (primer 1 SEQ ID NO:134, primer 2 SEQ ID NO:133),
AGR2 (primer 1 SEQ ID NO:2, primer 2 SEQ ID NO:1),
FOLH1 (primer 1 SEQ ID NO:29, primer 2 SEQ ID NO:28),
HOXDB13 (primer 1 SEQ ID NO:32, primer 2 SEQ ID NO:31),
KLK2 (primer 1 SEQ ID NO:35, primer 2 SEQ ID NO:34),
KLK3 (primer 1 SEQ ID NO:38, primer 2 SEQ ID NO:37), and
STEAP2 (primer 1 SEQ ID NO:125, primer 2 SEQ ID NO:124); and/or
wherein the breast cancer-selective genes are TFAP2C, S100A2, PGR, PIP, FAT2, AGR2, FAT1, RND3, PKP3, PRAME, and SCGB2A1,
wherein the primer sets corresponding to the breast cancer-selective genes are:
TFAP2C (primer 1 SEQ ID NO:131, primer 2 SEQ ID NO:130),
S100A2 (primer 1 SEQ ID NO:80, primer 79 SEQ ID NO:103),
PGR (primer 1 SEQ ID NO:62, primer 2 SEQ ID NO:61),
PIP (primer 1 SEQ ID NO:164, primer 2 SEQ ID NO:163),
FAT2 (primer 1 SEQ ID NO:26, primer 2 SEQ ID NO:25),
AGR2 (primer 1 SEQ ID NO:2, primer 2 SEQ ID NO:1),
FAT1 (primer 1 SEQ ID NO:23, primer 2 SEQ ID NO:22),
RND3 (primer 1 SEQ ID NO:77, primer 2 SEQ ID NO:76),
PKP3 (primer 1 SEQ ID NO:65, primer 2 SEQ ID NO:64),
PRAME (primer 1 SEQ ID NO:149, primer 2 SEQ ID NO:148), and
SCGB2A1 (primer 1 SEQ ID NO:83, primer 2 SEQ ID NO:82).

### Claim 2 (depends on 1)

The method of claim 1, further comprising reducing a volume of the product before isolating RNA.

### Claim 3 (depends on 1)

The method of claim 1, further comprising removing contaminants from the solution containing the cDNA molecules before encapsulating the cDNA molecules.

### Claim 4 (depends on 1)

The method of claim 1, wherein generating the cDNA molecules from the isolated RNA comprises conducting reverse transcription (RT) polymerase chain reaction (PCR) of the isolated RNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein amplifying cDNA within droplets comprises conducting PCR in a plurality of the droplets.

### Claim 6 (depends on 1)

The method of claim 1, wherein encapsulating individual cDNA further comprises encapsulating PCR reagents in individual droplets with the cDNA and forming at least 1000 droplets of a non-aqueous liquid.

### Claim 7 (depends on 1)

The method of claim 1, wherein the one or more reporter groups comprise a fluorescent label.

### Claim 8 (depends on 3)

The method of claim 3, wherein removing contaminants from the solution containing the cDNA molecules comprises the use of Solid Phase Reversible Immobilization (SPRI).

### Claim 9 (depends on 8)

The method of claim 8, wherein the SPRI comprises
immobilizing cDNA in the solution with magnetic beads that are configured to specifically bind to the cDNA;
removing contaminants from the solution; and
eluting purified cDNA.

### Claim 10 (depends on 6)

The method of claim 6, wherein the non-aqueous liquid comprises one or more fluorocarbons, hydrofluorocarbons, mineral oils, silicone oils, and hydrocarbon oils.

### Claim 11 (depends on 1)

The method of claim 1, wherein the CTCs arise from metastatic or primary/localized cancers.

### Claim 12 (depends on 1)

The method of claim 1, wherein analyzing the CTCs in the detected droplets comprises monitoring CTCs from blood samples taken over time from a patient with a known cancer, and testing, imaging, or both testing and imaging the CTCs to provide a prognosis for the patient.

### Claim 13 (depends on 1)

The method of claim 1, wherein analyzing the CTCs in the detected droplets comprises testing, imaging, or testing and imaging the CTCs to provide an indication of a response by the CTCs to a therapeutic intervention.

### Claim 14 (depends on 1)

The method of claim 1, wherein analyzing the CTCs in the detected droplets comprises determining a number or level of CTCs per unit volume of a blood sample from a patient to provide a measure of tumor burden in the patient.

### Claim 15 (depends on 14)

The method of claim 14, further comprising using the measure of tumor burden in the patient to select a therapy.

### Claim 16 (depends on 14)

The method of claim 14, further comprising determining the measure of tumor burden in the patient at a second time point to monitor the tumor burden over time.

### Claim 17 (depends on 1)

The method of claim 1, wherein the cDNA is pre-amplified prior to amplifying the cDNA within each of the droplet.

### Claim 18 (depends on 17)

The method of claim 17, wherein the cDNA is pre-amplified using nested primers corresponding to one or more primers that relate to one or more prostate cancer and/or breast cancer-selective genes.

### Claim 19 (depends on 17)

The method of claim 17, wherein the cDNA is pre-amplified using non-specific whole transcriptome amplification (WTA).