# US 12,215,392 B2 · Methods of preparing a DNA fraction enriched with circulating tumor DNA

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,215,392 B2
- Document ID: US12215392B2
- Status: Granted
- Kind: B2
- Application number: 18/633,462
- Filed: 2024-04-11
- Published: 2024-08-15
- Granted: 2025-02-04
- Earliest claimed: 2016-03-22
- Assignees: Myriad Women's Health, Inc.
- Applicants: Myriad Women's Health, Inc.
- Inventors: Jared Robert Maguire; Clement S. Chu; Imran Saeedul Haque; Eric Andrew Evans; Noah Welker
- Web page: https://patentgrep.com/patent/US12215392B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 35/0099: {comprising robots or similar manipulators (robots per se B25J)}
- C12Q 2600/156: Polymorphic or mutational markers
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure relates to a laboratory execution system that provides for automation of laboratory processes. A centralized data management system may be dynamically updated and used to facilitate management of components of the laboratory execution system, such as an automation system and an analytics results management system that may facilitate complex analytical functions, such as synthesizing raw test data. Potential workflows include the detection of specific molecules of interest.

## Claims

### Claim 1

A method for preparing a deoxyribonucleic acid (DNA) fraction enriched with circulating tumor DNA (ctDNA) from a cancer patient, comprising:
(a) obtaining a fluid sample taken from the cancer patient following completion of a cancer treatment, wherein the fluid sample comprises blood plasma or blood serum;
(b) extracting cell-free DNA (cfDNA) from the fluid sample;
(c) enriching a fraction of ctDNA by contacting the cfDNA with a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises at least 10 distinct oligonucleotides and each of the at least 10 distinct oligonucleotides comprises a nucleic acid sequence that is capable of hybridizing to a DNA fragment comprising a corresponding tumor-specific somatic mutation sequence selected from a predetermined set of tumor-specific somatic mutation sequences, wherein the predetermined set of tumor-specific somatic mutation sequences is was prepared by:
(i) obtaining a tumor sample and a non-tumor sample from the cancer patient;
(ii) sequencing genomic DNA from the tumor sample and sequencing genomic DNA from the non-tumor sample, thereby obtaining sequences of genomic DNA from the tumor sample and sequences of genomic DNA from the non-tumor sample;
(iii) aligning the sequences of genomic DNA from the tumor sample to a reference human genome that is not from the patient thereby obtaining a set of tumor-specific mutations and further obtaining from the set of tumor-specific mutations a set of tumor-specific somatic mutation sequences that are present in the sequences of genomic DNA from the tumor sample but not present in the sequences of genomic DNA from the non-tumor sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein enriching the fraction of cfDNA corresponding to ctDNA comprises:
(i) hybrid capture-based enrichment;
(ii) PCR-target enrichment; or
(iii) on-sequencer enrichment.

### Claim 3 (depends on 1)

The method of claim 1, wherein sequencing genomic DNA from the tumor sample of the patient and sequencing genomic DNA from the non-tumor sample of the patient comprises whole genome sequencing or targeted sequencing of exomes.

### Claim 4 (depends on 1)

The method of claim 1, further comprising:
sequencing the ctDNA, thereby obtaining a plurality of sequence reads; and
detecting the presence or absence of a DNA fragment comprising any one of the set of tumor-specific somatic mutation sequences.

### Claim 5 (depends on 1)

The method of claim 1, wherein the cancer patient has a cancer selected from adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain/CNS tumor, breast cancer, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

### Claim 6 (depends on 1)

The method of claim 1, wherein the plurality of oligonucleotides comprises at least 50 distinct oligonucleotides capable of detecting at least 50 different tumor-specific somatic mutations.

### Claim 7

A method for preparing a deoxyribonucleic acid (DNA) fraction enriched with circulating tumor DNA (ctDNA) from a cancer patient, comprising:
(a) obtaining a fluid sample taken from the cancer patient while the cancer patient is in remission, wherein the fluid sample comprises blood plasma or blood serum;
(b) extracting cell-free DNA (cfDNA) from the fluid sample;
(c) enriching a fraction of ctDNA by contacting the cfDNA with a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises at least 10 distinct oligonucleotides and each of the at least 10 distinct oligonucleotides comprises a nucleic acid sequence that is capable of hybridizing to a DNA fragment comprising a corresponding tumor-specific somatic mutation sequence selected from a predetermined set of tumor-specific somatic mutation sequences, wherein the predetermined set of tumor-specific somatic mutation sequences is was prepared by:
(i) obtaining a tumor sample and a non-tumor sample from the cancer patient;
(ii) sequencing genomic DNA from the tumor sample and sequencing genomic DNA from the non-tumor sample, thereby obtaining sequences of genomic DNA from the tumor sample and sequences of genomic DNA from the non-tumor sample;
(iii) aligning the sequences of genomic DNA from the tumor sample to a reference human genome that is not from the patient thereby obtaining a set of tumor-specific mutations and further obtaining from the set of tumor-specific mutations a set of tumor-specific somatic mutation sequences that are present in the sequences of genomic DNA from the tumor sample but not present in the sequences of genomic DNA from the non-tumor sample.

### Claim 8 (depends on 7)

The method of claim 7, wherein enriching the fraction of cfDNA corresponding to ctDNA comprises:
(i) hybrid capture-based enrichment;
(ii) PCR-target enrichment; or
(iii) on-sequencer enrichment.

### Claim 9 (depends on 7)

The method of claim 7, wherein sequencing genomic DNA from the tumor sample of the patient and sequencing genomic DNA from the non-tumor sample of the patient comprises whole genome sequencing or targeted sequencing of exomes.

### Claim 10 (depends on 7)

The method of claim 7, further comprising:
sequencing the ctDNA, thereby obtaining a plurality of sequence reads; and
detecting the presence or absence of a DNA fragment comprising any one of the set of tumor-specific somatic mutation sequences.

### Claim 11 (depends on 7)

The method of claim 7, wherein the cancer patient has a cancer selected from adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain/CNS tumor, breast cancer, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

### Claim 12 (depends on 7)

The method of claim 7, wherein the plurality of oligonucleotides comprises at least 50 distinct oligonucleotides capable of detecting at least 50 different tumor-specific somatic mutations.

### Claim 13

A method for preparing a deoxyribonucleic acid (DNA) fraction enriched with circulating tumor DNA (ctDNA) from a cancer patient, comprising:
(a) obtaining a fluid sample taken from the cancer patient at a time coinciding with or prior to surgery; following, during, or prior to administration of chemotherapy; or following, during, or prior to radiation therapy; wherein the fluid sample comprises blood plasma or blood serum;
(b) extracting cell-free DNA (cfDNA) from the fluid sample;
(c) enriching a fraction of ctDNA by contacting the cfDNA with a plurality of oligonucleotides, wherein the plurality of oligonucleotides comprises at least 10 distinct oligonucleotides and each of the at least 10 distinct oligonucleotides comprises a nucleic acid sequence that is capable of hybridizing to a DNA fragment comprising a corresponding tumor-specific somatic mutation sequence selected from a predetermined set of tumor-specific somatic mutation sequences, wherein the predetermined set of tumor-specific somatic mutation sequences is was prepared by:
(i) obtaining a tumor sample and a non-tumor sample from the cancer patient;
(ii) sequencing genomic DNA from the tumor sample and sequencing genomic DNA from the non-tumor sample, thereby obtaining sequences of genomic DNA from the tumor sample and sequences of genomic DNA from the non-tumor sample;
(iii) aligning the sequences of genomic DNA from the tumor sample to a reference human genome that is not from the patient thereby obtaining a set of tumor-specific mutations and further obtaining from the set of tumor-specific mutations a set of tumor-specific somatic mutation sequences that are present in the sequences of genomic DNA from the tumor sample but not present in the sequences of genomic DNA from the non-tumor sample.

### Claim 14 (depends on 13)

The method of claim 13, wherein enriching the fraction of cfDNA corresponding to ctDNA comprises:
(i) hybrid capture-based enrichment;
(ii) PCR-target enrichment; or
(iii) on-sequencer enrichment.

### Claim 15 (depends on 13)

The method of claim 13, wherein sequencing genomic DNA from the tumor sample of the patient and sequencing genomic DNA from the non-tumor sample of the patient comprises whole genome sequencing or targeted sequencing of exomes.

### Claim 16 (depends on 13)

The method of claim 13, further comprising:
sequencing the ctDNA, thereby obtaining a plurality of sequence reads; and
detecting the presence or absence of a DNA fragment comprising any one of the set of tumor-specific somatic mutation sequences.

### Claim 17 (depends on 13)

The method of claim 13, wherein the cancer patient has a cancer selected from adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain/CNS tumor, breast cancer, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

### Claim 18 (depends on 13)

The method of claim 13, wherein the fluid sample is taken from the cancer patient at a time coinciding with or prior to surgery.

### Claim 19 (depends on 13)

The method of claim 13, wherein the fluid sample is taken from the cancer patient at a time following, during, or prior to administration of chemotherapy.

### Claim 20 (depends on 13)

The method of claim 13, wherein the fluid sample is taken from the cancer patient at a time following, during, or prior to radiation therapy.

### Claim 21 (depends on 1)

The method of claim 1, further comprising repeating (a)-(c) one or more times.

### Claim 22 (depends on 7)

The method of claim 7, further comprising repeating (a)-(c) one or more times.

### Claim 23 (depends on 13)

The method of claim 13, further comprising repeating (a)-(c) one or more times.