	US 12,410,480 B2
	Methods and systems for detecting colorectal cancer via nucleic acid methylation analysis
John St. John, Brisbane, CA (US); Steven Kothen-Hill, Millbrae, CA (US); Rui Yang, Burlingame, CA (US); and Adam Drake, Pacifica, CA (US)
Assigned to Freenome Holdings, Inc., San Francisco, CA (US)
Filed by Freenome Holdings, Inc., South San Francisco, CA (US)
Filed on Feb. 1, 2023, as Appl. No. 18/163,138.
Application 18/163,138 is a continuation of application No. 17/954,576, filed on Sep. 28, 2022.
Application 17/954,576 is a continuation of application No. PCT/US2021/024604, filed on Mar. 29, 2021.
Claims priority of provisional application 63/002,878, filed on Mar. 31, 2020.
Prior Publication US 2023/0220492 A1, Jul. 13, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6869 (2018.01); G16B 20/10 (2019.01); G16B 40/20 (2019.01)

CPC C12Q 1/6886 (2013.01) [G16B 20/10 (2019.02); G16B 40/20 (2019.02); C12Q 1/6806 (2013.01); C12Q 1/6869 (2013.01); C12Q 2600/154 (2013.01)]

13 Claims

1. A method of treating a colorectal cancer in a subject, the method comprising:

(a) subjecting a cell-free deoxyribonucleic acid (cfDNA) sample obtained or derived from the subject to conditions sufficient to convert unmethylated cytosines to uracils in nucleic acid molecules of the cfDNA sample, thereby generating a plurality of converted nucleic acid molecules;

(b) contacting the plurality of converted nucleic acid molecules with nucleic acid probes complementary to a pre-determined methylation signature panel of at least two genomic regions selected from the group consisting of

ITGA4, chr2: 181457004-181457950;

EMBP1, chr1: 121519076-121519744;

TMEM163, chr2: 134718243-134719428;

SFMBT2, chr10: 7408046-7408953;

ELMO1, chr7: 37448612-37449471;

ZNF543, chr19: 57320164-57320845;

SFMBT2, chr10: 7410025-7411008;

CHST10, chr2: 100417269-100417795;

ELMO1, chr7: 37447852-37448217;

CCNA1, chr13: 36431498-36432414;

BEND4, chr4: 42150707-42153216;

KRBA1, chr7: 149714695-149715338;

S1PR1, chr1: 101236505-101237190;

PPP1R16B, chr20: 38805341-38807221;

IKZF1, chr7: 50304053-50304994;

LONRF2, chr2: 100322082-100322599;

ZFP82, chr19: 36418330-36418931;

FLT3, chr13: 28099881-28100943;

FBN1, chr15: 48644595-48646444; and

FLI1, chr11: 128693042-128694372,

to enrich for sequences corresponding to the pre-determined methylation signature panel;

(c) determining a threshold value for each of the at least two genomic regions, wherein the threshold value is indicative of a number of methylated CpG sites in each of the at least two genomic regions;

(d) comparing the number of methylated CpG sites in each of the at least two genomic regions to the threshold value;

(e) determining that the number of methylated CpG sites in each of the at least two genomic regions is above the threshold value, thereby identifying the subject as having the colorectal cancer; and

(f) responsive to the determining in (e), administering a therapeutic intervention to the subject to treat the colorectal cancer in the subject, wherein the therapeutic intervention comprises a chemotherapy, a radiotherapy, an immunotherapy, or a surgery.