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US-20260125763-A1 - METHODS AND SYSTEMS FOR CELL-FREE NUCLEIC ACID PROCESSING

US20260125763A1US 20260125763 A1US20260125763 A1US 20260125763A1US-20260125763-A1

Abstract

Methods and systems for targeted detection of circulating tumor DNA (ctDNA) molecules are disclosed herein. In some cases, a molecular sequencing library depleted of methylated DNA can be generated and used to detect ctDNA in a cell-free DNA sample reliably at a lower sequencing depth and lower cost than existing methods.

Inventors

  • Daniel Diniz De Carvalho
  • Abel Licon
  • Yulia NEWTON
  • Jun Min
  • Iulia CIRLAN
  • Shu Yi SHEN
  • Felicia VINCELLI
  • Junjun Zhang

Assignees

  • ADELA, INC.

Dates

Publication Date
20260507
Application Date
20251009

Claims (20)

  1. 1 - 184 . (canceled)
  2. 185 . A method for detecting minimal residual disease (MRD), comprising: (a) assaying a biological sample from a subject, wherein said assaying does not comprise analyzing a solid tumor sample of the subject, wherein said assaying comprises sequencing of one or more enriched methylated regions in said biological sample; and (b) detecting said MRD at a specificity of at least 90% or at a sensitivity of at least 80% based at least in part on said sequencing of said one or more methylated regions.
  3. 186 . The method of claim 185 , wherein said biological sample is a plasma or blood sample.
  4. 187 . The method of claim 185 , wherein said biological sample comprises a plurality of methylated nucleic acid molecules.
  5. 188 . The method of claim 185 , wherein said biological sample has no more than 50 ng of nucleic acid molecules.
  6. 189 . The method of claim 187 , wherein said assaying comprises enriching for said plurality of methylated nucleic acid molecules using a capture reagent, thereby generating a plurality of enriched nucleic acid molecules.
  7. 190 . The method of claim 189 , wherein said enriching is under conditions sufficient to increase a fold enrichment ratio associated with said plurality of methylated nucleic acid molecules.
  8. 191 . The method of claim 189 , wherein said capture reagent comprises a binder.
  9. 192 . The method of claim 191 , wherein said binder comprises a Methyl-CpG-binding domain.
  10. 193 . The method of claim 188 , wherein said enriching comprises immunoprecipitating said plurality of methylated nucleic acid molecules using an antibody.
  11. 194 . The method of claim 189 , further comprising contacting said plurality of enriched nucleic acids with one or more nucleic acid capture probes to enrich for one or more target sequences.
  12. 195 . The method of claim 194 , wherein said one or more nucleic acid capture probes comprises sequences associated with healthy samples.
  13. 196 . The method of claim 187 , further comprising amplifying said plurality of methylated nucleic acids to generate a plurality of amplified nucleic acids.
  14. 197 . The method of claim 185 , wherein said sequencing of said one or more methylated regions comprises unbiased sequencing or targeted sequencing.
  15. 198 . The method of claim 185 , comprising detecting said MRD at a specificity of at least 95% or at a sensitivity of at least 90%.
  16. 199 . The method of claim 185 , comprising detecting said MRD at an AUROC of at least about 90%.
  17. 200 . The method of claim 185 , further comprising processing sequencing reads of said one or more methylated regions to generate a methylation profile of said biological sample.
  18. 201 . The method of claim 200 , wherein said processing comprises using a machine-learning derived classifier.
  19. 202 . The method of claim 201 , wherein said machine-learning derived classifier comprises an elastic net classifier, lasso, support vector machine, random forest, or neural network.
  20. 203 . The method of claim 200 , wherein said methylation profile comprises whole methylome.

Description

CROSS REFERENCE This application is a continuation of International Application No. PCT/US2024/024491, filed Apr. 12, 2024, which claims the benefit of U.S. Provisional Application No. 63/496,347, filed Apr. 14, 2023, U.S. Provisional Application No. 63/501,359, filed May 10, 2023, U.S. Provisional Application No. 63/511,441, filed Jun. 30, 2023, U.S. Provisional Application No. 63/517,327, filed Aug. 2, 2023, U.S. Provisional Application No. 63/588,120 filed Oct. 5, 2023, U.S. Provisional Application No. 63/591,732, filed Oct. 19, 2023, U.S. Provisional Application No. 63/594,365, filed Oct. 30, 2023, U.S. Provisional Application No. 63/602,156, filed Nov. 22, 2023, U.S. Provisional Application No. 63/549,294, filed Feb. 2, 2024, and U.S. Provisional Application No. 63/571,139 filed Mar. 28, 2024, each of which are incorporated herein by reference in their entirety. BACKGROUND Circulating tumor DNA (ctDNA) has increasingly demonstrated potential as a non-invasive, tumor-specific biomarker for routine clinical use. ctDNA is derived from tumor cells predominantly undergoing cell-death and released into circulation of various bodily fluids including blood. In most cancer patients, the majority of blood-derived cell-free DNA originates from healthy (e.g., non-cancerous) tissues. In addition, the fraction of ctDNA observed may range from <0.1% to 90% of total cell-free DNA at diagnosis depending on several factors including primary site of the tumor and disease burden. ctDNA provides non-invasive access to the tumor's molecular landscape and disease burden. INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. SUMMARY In some aspects, provided herein is a method, comprising: (a) providing a plurality of nucleic acid molecules generated from a cell-free deoxynucleic acid (cfDNA) sample of a subject; (b) subjecting said plurality of nucleic acid molecules or derivatives thereof to sequencing to generate a plurality of sequencing reads; (c) computer processing said plurality of sequencing reads generate a methylation profile for said plurality of nucleic acid molecules; and (d) computer processing said methylation profile to determine that said subject has cancer at an area under the receiver operating characteristic curve (AUROC) of at least about 91%, wherein said cancer is low-shedding cancer. In some embodiments, said cfDNA sample comprises circulating tumor nucleic acid molecules derived from a low-shedding tumor. In some embodiments, said cancer is bladder cancer, breast cancer, endometrial cancer, prostate cancer, or renal cancer. In some embodiments, said cancer is endometrial cancer or prostate cancer. In some aspects, provided herein, is a method, comprising: (a) providing a plurality of nucleic acid molecules generated from a cell-free deoxynucleic acid (cfDNA) sample of a subject; (b) subjecting said plurality of nucleic acid molecules or derivatives thereof to sequencing to generate a plurality of sequencing reads; (c) computer processing said plurality of sequencing reads generate a methylation profile for said plurality of nucleic acid molecules; and (d) computer processing said methylation profile to determine that said subject has cancer at an area under the receiver operating characteristic curve (AUROC) of at least about 94% and wherein said cancer is an early-stage cancer. In some embodiments, said cfDNA sample comprises circulating tumor nucleic acid molecules derived from an early-stage tumor. In some embodiments, said early-stage tumor is stage I tumor. In some embodiments, said early-stage tumor is stage II tumor. In some embodiments, said cancer is bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head & neck cancer, hepatobiliary cancer, lung cancer, ovarian cancer, prostate cancer, or renal cancer. In some embodiments, said cancer is esophageal cancer, hepatobiliary cancer, or ovarian cancer. In some aspects, provided herein, is a method, comprising: (a) providing a plurality of nucleic acid molecules generated from a cell-free deoxynucleic acid (cfDNA) sample of a subject; (b) subjecting said plurality of nucleic acid molecules or derivatives thereof to sequencing to generate a plurality of sequencing reads in absence of bisulfite conversion; (c) computer processing said plurality of sequencing reads generate a methylation profile for said plurality of nucleic acid molecules; and (d) computer processing said methylation profile to determine that said subject has cancer, wherein said cancer is endometrial cancer, esophageal cancer, hepatobiliary cancer, ovarian cancer, prostate cancer, bladder cancer, breast cancer, colorectal cancer, head and neck cancer, lung cancer, pancreatic cancer, or renal can