# US 2019/0169685 A1 · Process for microsatellite instability detection

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- Number: US 2019/0169685 A1
- Document ID: US20190169685A1
- Status: Published application
- Kind: A1
- Application number: 16/204,642
- Filed: 2018-11-29
- Published: 2019-06-06
- Earliest claimed: 2017-12-01
- Assignees: Personal Genome Diagnostics Inc.
- Applicants: Personal Genome Diagnostics Inc.
- Inventors: Andrew Georgiadis; Mark Sausen
- Web page: https://patentgrep.com/patent/US20190169685A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G16B 5/20: Probabilistic models
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16H 10/40: for data related to laboratory analysis, e.g. patient specimen analysis
- G16H 15/00: ICT specially adapted for medical reports, e.g. generation or transmission thereof
- G16H 20/00: ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
- G16H 50/20: for computer-aided diagnosis, e.g. based on medical expert systems
- G16H 50/30: for calculating health indices; for individual health risk assessment
- C12Q 2563/185: Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
- 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
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA

## Abstract

The invention provides methods for determining the MSI status of a patient by liquid biopsy with sample preparation using hybrid capture and non-unique barcodes. In certain aspects, the invention provides a method of detecting microsatellite instability (MSI). The method includes obtaining cell-free DNA (cfDNA) from a sample of blood or plasma from a patient and sequencing portions of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA. A report is provided describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution.

## Claims

### Claim 1

A method of determining micro satellite instability (MSI) comprising:
obtaining cell-free DNA (cfDNA) from a liquid sample from a patient;
sequencing fragments of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA; and
providing a report describing an MSI status of the patient depicting a distribution of lengths of the plurality of tracts that have peaks that deviate from peaks in a reference distribution.

### Claim 2 (depends on 1)

The method of claim 1, wherein the reference distribution is from a matched normal DNA sample.

### Claim 3 (depends on 2)

The method of claim 2, further comprising attaching non-unique barcodes to the fragments.

### Claim 4 (depends on 3)

The method of claim 3, further comprising amplifying the fragments to produce amplicons that include barcode information and copies of the fragments, wherein the sequencing step comprises sequencing the amplicons.

### Claim 5 (depends on 4)

The method of claim 4, wherein the obtained sequences include a plurality of sequence reads and the method includes: aligning the sequence reads to a reference, and identifying groups of sequence reads that originated from a unique cfDNA fragment by means of the barcode information and position or content of the sequence reads.

### Claim 6 (depends on 5)

The method of claim 5, wherein the use of the non-unique barcodes to identify groups of sequence reads that originated from a unique cfDNA fragment which allows for the lengths of the plurality of tracts to be determined correctly by correcting for errors introduced by sequencing artifacts or polymerase slippage during the amplifying step.

### Claim 7 (depends on 4)

The method of claim 4, wherein the target portions are markers for MSI.

### Claim 8 (depends on 7)

The method of claim 7, wherein the markers include one or more of BAT25, BAT26, MON027, NR21, NR24, Penta C, and Penta D.

### Claim 9 (depends on 7)

The method of claim 7, wherein the markers include BAT25, BAT26, MON027, NR21, and NR24.

### Claim 10 (depends on 7)

The method of claim 7, wherein the one or more microsatellite markers are selected from the group consisting of BAT-25, BAT-26, MONO-27, NR-21, NR-24, Penta C, and Penta D.

### Claim 11 (depends on 7)

The method of claim 7, further comprising recommending a treatment for the patient based on the MSI status.

### Claim 12 (depends on 11)

The method of claim 11, wherein the MSI status indicates that the patient is microsatellite instable and the treatment comprises an immune checkpoint inhibitor.

### Claim 13 (depends on 12)

The method of claim 12, further comprising administering the immune checkpoint inhibitor to the patient.

### Claim 14 (depends on 13)

The method of claim 13, wherein the immune checkpoint inhibitor comprises an antibody.

### Claim 15 (depends on 14)

The method of claim 14, wherein the antibody is selected from the group consisting of: an anti-PD-1 antibody; an anti-IDO antibody; anti-CTLA-4 antibody; an anti-PD-L1 antibody; and an anti-LAG-3 antibody.

### Claim 16 (depends on 1)

The method of claim 1, wherein the sequencing is next-generation, short-read sequencing.

### Claim 17 (depends on 1)

The method of claim 1, wherein the sample is from blood, serum, plasma, urine, saliva or other biological fluid.

### Claim 18

A method of detecting microsatellite instability (MSI), the method comprising:
obtaining a sample comprising fragments of cell-free DNA (cfDNA) from a patient;
attaching barcodes to the fragments, wherein at least some of the barcodes are not unique;
sequencing the barcodes to obtain sequences of a plurality of markers in the DNA;
determining a distribution of lengths of the plurality of markers;
providing a report describing MSI in the patient when peaks in the distribution deviate from expected peaks in a modeled healthy or reference distribution.

### Claim 19

A method of detecting micro satellite instability (MSI), the method comprising:
obtaining fragments cell-free DNA (cfDNA) from a sample of plasma from a patient; sequencing portions of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA;
assembling the sequences using information from endogenous barcodes at the starts and ends of the fragments; and
providing a report describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution.

### Claim 20 (depends on 7)

The method of claim 7, wherein the markers comprise any polynucleotide tract.

### Claim 21

The method of any of the preceding claims, wherein the method is used in screening for Lynch syndrome.

### Claim 22 (depends on 1)

A method for generating a personalized cancer treatment report, the method comprising: acquiring a liquid sample from a subject having or suspected of having cancer, determining the MSI status of the subject by the method of claim 1 and generating a personalized cancer treatment report to memorialize the presence or absence of microsatellite instability in the subject.

### Claim 23 (depends on 22)

The method of claim 22, wherein the cancer treatment report comprises one or more of the following: (i) information on prognosis, resistance, or potential therapeutic options; (ii) information on the likely effectiveness of a therapeutic option; (iii) the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to the subject; or (iv) information on the administration of a drug.

### Claim 24 (depends on 22)

The method of claim 22, wherein the sample comprises cfDNA.

### Claim 25 (depends on 1)

The method of claim 1, wherein the subject has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers.

### Claim 26 (depends on 18)

The method of claim 18, wherein the subject has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers.

### Claim 27 (depends on 19)

The method of claim 19, wherein the subject has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers.

### Claim 28 (depends on 22)

The method of claim 22, wherein the subject has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers.

### Claim 29 (depends on 18)

A method for generating a personalized cancer treatment report, the method comprising: acquiring a liquid sample from a subject having or suspected of having cancer, determining the MSI status of the subject by the method of claim 18 and generating a personalized cancer treatment report to memorialize the presence or absence of microsatellite instability in the subject.

### Claim 30 (depends on 19)

A method for generating a personalized cancer treatment report, the method comprising: acquiring a liquid sample from a subject having or suspected of having cancer, determining the MSI status of the subject by the method of claim 19 and generating a personalized cancer treatment report to memorialize the presence or absence of microsatellite instability in the subject.