# US 12,110,560 B2 · Methods for monitoring residual disease

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- Number: US 12,110,560 B2
- Document ID: US12110560B2
- Status: Granted
- Kind: B2
- Application number: 18/594,336
- Filed: 2024-03-04
- Published: 2024-06-20
- Granted: 2024-10-08
- Earliest claimed: 2012-09-04
- Assignees: Guardant Health, Inc.
- Applicants: Guardant Health, Inc.
- Inventors: AmirAli Talasaz
- Web page: https://patentgrep.com/patent/US12110560B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The present disclosure provides a method for monitoring residual disease in a subject. Generally, the method comprises determining a frequency of cancer mutations from a first sample obtained from a tumor biopsy from the subject; determining a frequency of the cancer mutations discovered in the first sample from a second sample comprising cell-free deoxyribonucleic acid (cfDNA) molecules that is obtained from the subject after the subject has undergone a course of treatment for cancer; and determining a presence or absence of cancer in the subject based on an analysis of the frequency of the cancer mutations from sequence data from the second sample.

## Claims

### Claim 1

A method for monitoring residual disease in a subject, the method comprising:
(a) providing a first sample from the subject comprising genomic polynucleotides and sequencing the genomic polynucleotides or amplicons thereof to generate a first set of sequence data, wherein the first sample is obtained from a tumor biopsy from the subject;
(b) determining a frequency of cancer mutations from the first set of sequence data from the first sample;
(c) providing a second sample from the subject comprising cell-free deoxyribonucleic acid (cfDNA) molecules and sequencing the cfDNA molecules or amplicons thereof to produce a second set of sequence data, wherein the second sample is obtained from the subject after the subject has undergone a course of treatment for cancer;
(d) determining a frequency of the cancer mutations discovered in the first sample from the second set of sequence data from the second sample; and
(e) determining a presence or absence of cancer in the subject based on an analysis of the frequency of the cancer mutations from the second set of sequence data from the second sample, thereby monitoring for the residual disease in the subject.

### Claim 2 (depends on 1)

The method of claim 1, wherein the second sample is a blood sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein the second sample comprises between 1 nanogram (ng) to 100 ng of cfDNA molecules.

### Claim 4 (depends on 2)

The method of claim 2, wherein adapters are ligated to a plurality of the cfDNA molecules prior to sequencing to generate tagged polynucleotides.

### Claim 5 (depends on 4)

The method of claim 4, wherein the adapters comprise binding sites for universal amplification primers.

### Claim 6 (depends on 4)

The method of claim 4, wherein the tagged polynucleotides are amplified to generate amplified progeny polynucleotides.

### Claim 7 (depends on 6)

The method of claim 6, wherein the amplified progeny polynucleotides are enriched for target regions of interest.

### Claim 8 (depends on 7)

The method of claim 7, wherein the enrichment comprises an amplification-based enrichment.

### Claim 9 (depends on 8)

The method of claim 8, wherein the target regions of interest comprise regions associated with cancer.

### Claim 10 (depends on 9)

The method of claim 9, wherein the cancer is colorectal cancer, lung cancer, or breast cancer.

### Claim 11 (depends on 8)

The method of claim 8, wherein the first set of sequence data and the second set of sequence data comprise sequencing reads.

### Claim 12 (depends on 11)

The method of claim 11, wherein the sequencing reads from the second set of sequencing data from the second sample are mapped to a reference sequence.

### Claim 13 (depends on 12)

The method of claim 12, wherein a plurality of sequencing reads that map to the reference sequence are grouped into families having a same start and stop base position.

### Claim 14 (depends on 12)

The method of claim 12, wherein the adapters further comprise molecular barcodes, and wherein a plurality of sequencing reads that map to the reference sequence are grouped into families having a same start and stop base position and the same molecular barcode sequence.

### Claim 15 (depends on 12)

The method of claim 12, wherein cancer mutations in the second set of sequence data from the second sample are determined by comparing the sequencing reads to the reference sequence.

### Claim 16 (depends on 13)

The method of claim 13, wherein cancer mutations in the second set of sequence data from the second sample are determined by collapsing sequencing reads within each family to yield a base call at a genetic locus and determining a frequency of one or more bases called at the locus from among the families.

### Claim 17 (depends on 14)

The method of claim 14, wherein cancer mutations in the second set of sequence data from the second sample are determined by collapsing sequencing reads within each family to yield a base call at a genetic locus and determining a frequency of one or more bases called at the locus from among the families.

### Claim 18 (depends on 1)

The method of claim 1, wherein the first set of sequence data comprises sequencing reads from the genomic polynucleotides, wherein the frequency of cancer mutations from the first set of sequence data from the first sample is determined by comparing sequencing reads to a reference sequence.

### Claim 19 (depends on 1)

The method of claim 1, wherein the course of treatment is selected based on the frequency of cancer mutations determined from the first sample.

### Claim 20 (depends on 14)

The method of claim 14, wherein the plurality of cfDNA molecules is ligated to adapters comprising n number of molecular barcodes, wherein n is at least 2 and no more than 10,000*z, wherein z is a mean of an expected number of duplicate molecules of cfDNA molecules in the second sample having identical start and stop positions.