	US 11,898,209 B2
	Digital analysis of circulating tumor cells in blood samples
Daniel A. Haber, Chestnut Hill, MA (US); Ravi Kapur, Sharon, MA (US); Mehmet Toner, Charlestown, MA (US); Shyamala Maheswaran, Lexington, MA (US); Xin Hong, Medford, MA (US); David Tomoaki Miyamoto, Wellesley, MA (US); Tanya Todorova, Malden, MA (US); and Sarah Javaid, Cambridge, MA (US)
Assigned to The General Hospital Corporation, Boston, MA (US)
Filed by The General Hospital Corporation, Boston, MA (US)
Filed on Oct. 8, 2020, as Appl. No. 17/065,889.
Application 17/065,889 is a continuation of application No. 15/560,324, abandoned, previously published as PCT/US2016/024367, filed on Mar. 25, 2016.
Claims priority of provisional application 62/253,619, filed on Nov. 10, 2015.
Claims priority of provisional application 62/219,339, filed on Sep. 16, 2015.
Claims priority of provisional application 62/137,891, filed on Mar. 25, 2015.
Prior Publication US 2021/0189501 A1, Jun. 24, 2021
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/686 (2018.01)

CPC C12Q 1/6886 (2013.01) [C12Q 1/686 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01)]

19 Claims

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).