US 12,247,259 B2
Using nucleic acid size range for noninvasive cancer detection
Yuk-Ming Dennis Lo, Homantin (CN); Rossa Wai Kwun Chiu, Shatin (CN); Kwan Chee Chan, Mei Foo Sun Chuen (CN); and Peiyong Jiang, Pak Shek Kok (CN)
Assigned to The Chinese University of Hong Kong, New Territories (HK); and GRAIL, Inc., Menlo Park, CA (US)
Filed by The Chinese University of Hong Kong, Shatin (HK); and GRAIL, Inc., Menlo Park, CA (US)
Filed on Nov. 8, 2021, as Appl. No. 17/521,333.
Application 17/521,333 is a division of application No. 16/178,378, filed on Nov. 1, 2018, granted, now 11,168,356.
Claims priority of provisional application 62/580,906, filed on Nov. 2, 2017.
Prior Publication US 2022/0064714 A1, Mar. 3, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6809 (2018.01); C12Q 1/6883 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6883 (2013.01); C12Q 1/6809 (2013.01); C12Q 2600/154 (2013.01); C12Q 2600/156 (2013.01)] 23 Claims
 
1. A method of determining a cancer classification in a biological sample from a subject, wherein the biological sample includes a mixture of cell-free DNA molecules including tumor DNA molecules and non-tumor DNA molecules, wherein the biological sample comprises plasma or serum, the method comprising:
performing a physical size separation of the biological sample for a range of sizes of the cell-free DNA molecules to form an enriched biological sample;
performing methylation-aware sequencing to identify methylated cell-free DNA molecules in the enriched biological sample;
for each size range of a plurality of size ranges:
measuring a first amount of the methylated cell-free DNA molecules from the enriched biological sample corresponding to the size range, and
determining, by a computer system, a methylation level using the first amount of the methylated cell-free DNA molecules corresponding to the size range and a second amount of DNA molecules in a second size range that includes sizes not in the size range;
obtaining, by the computer system, a reference size pattern including a reference methylation level for each size range of the plurality of size ranges, wherein the reference size pattern is determined from a plurality of reference samples from subjects with cancer or from subjects without cancer;
comparing, by the computer system, a plurality of the methylation levels to the reference size pattern; and
determining a level of cancer based on the comparison.