# US 11,993,819 B2 · Method of determining a risk of cancer

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- Number: US 11,993,819 B2
- Document ID: US11993819B2
- Status: Granted
- Kind: B2
- Application number: 16/977,197
- Filed: 2019-03-01
- Published: 2021-05-27
- Granted: 2024-05-28
- Earliest claimed: 2018-03-01
- Assignees: Agency for Science, Technology and Research
- Applicants: Agency for Science, Technology and Research
- Inventors: Anders Skanderup; Yu Amanda Guo; Sarah Ng
- Web page: https://patentgrep.com/patent/US11993819B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/6827: for detection of mutation or polymorphism
- 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

## Abstract

There is provided a method of determining a risk of gastrointestinal cancer in a human subject, the method comprising: determining in a biological sample of the subject, whether mutation is present in at least one CTCF-binding sites (CBS) overlapping regions, or portions or flanking sequences thereof, or at least one non-CBS regions, or portions or flanking sequences thereof, wherein presence of mutation in at least one CBS overlapping regions, or portions or flanking sequences thereof, and/or at least one non-CBS regions, or portions or flanking sequences thereof, is indicative of a risk of gastrointestinal cancer in the subject. There are also provided a method of treating gastrointestinal cancer in a human subject and related kits.

## Claims

### Claim 1

A method of detecting, determining and/or diagnosing gastrointestinal cancer in a human subject, the method comprising:
i) extracting nucleic acid from a fluid biological sample of the subject, wherein the fluid biological sample is selected from the group consisting of blood, plasma, serum, and combinations thereof,
ii) contacting the nucleic acid from the sample with an agent for detecting the presence of a mutation in one or more focal regions with high mutation rates,
iii) detecting the presence of mutation in one or more focal regions with high mutation rates,
wherein the one or more focal regions is selected from the group consisting of CTCF-binding sites (CBS) overlapping regions set forth in the table 1 below:
Chromosome
Start site
End site
6
50570094
50570120
8
71000992
71001012
1
209422184
209422222
2
49173770
49173816
4
182064578
182064613
X
104435106
104435140
14
70285576
70285601
6
73122084
73122123
8
70576141
70576184
13
36552821
36552860
3
164903700
164903728
or portions or 1 to 4 bp sequences flanking the 5′ end thereof,
and non-CBS regions set forth in the table 2 below:
Chromosome
Start site
End site
7
68391104
68391132
7
136495924
136495948
2
57627616
57627640
16
8381278
8381302
5
23824204
23824224
7
67614923
67614943
8
65161396
65161420
7
4937707
4937736
12
126996666
126996686
4
5415060
5415082
X
137405623
137405655
3
171164993
171165017
4
144748744
144748764
9
25481736
25481758
2
77150455
77150477
3
104801455
104801477
X
125548690
125548710
14
83046706
83046744
or portions or 1 to 4 bp sequences flanking the 5′ end thereof, and
wherein detecting the presence of mutation in the CBS overlapping region(s), or portions or 1 to 4 bp sequences flanking the 5′ end thereof, comprises contacting the biological sample with a primer having a sequence selected from the group consisting of: SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. SEQ ID NO. 83, SEQ ID NO. 84 and combinations thereof and/or wherein detecting the presence of mutation in the one or more focal regions comprises contacting the biological sample with a primer having a sequence selected from the group consisting of: SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, and combinations thereof; and
iv) detecting, determining, and/or diagnosing the presence of gastrointestinal cancer in the subject based on the presence of mutation in the one or more focal regions.

### Claim 2 (depends on 1)

The method of claim 1, further comprising determining in the fluid biological sample of the subject, whether mutation is present in at least one of the genes selected from the group consisting of: KRAS, TP53, RHOA, ARID1A, PIK3CA, ERBB3 and PTEN, wherein presence of mutation in at least one of the genes is indicative of a risk of gastrointestinal cancer in the subject.

### Claim 3 (depends on 1)

The method of claim 1, wherein detecting the presence of mutation in the one or more focal regions does not comprise determining whether mutation is present in a region spanning more than 50 nucleotides.

### Claim 4 (depends on 1)

The method of claim 1, wherein the method further comprises determining whether mutation is present in at least two of the CBS overlapping regions, or portions or flanking sequences thereof, set forth in the table 1, optionally wherein the method comprises determining whether mutation is present in all of the CBS overlapping regions, or portions or 1 to 4 bp sequences flanking the 5′ end thereof, set forth in the table 1, further optionally wherein the method comprises determining whether mutation is present in all of the CBS overlapping regions, or portions or 1 to 4 bp sequences flanking the 5′ end thereof, and all of the non-CBS regions, or portions or 1 to 4 bp sequences flanking the 5′ end thereof, set forth in the tables 1 and 2.

### Claim 5 (depends on 1)

The method of claim 1, wherein the gastrointestinal cancer is selected from the group consisting of gastric cancer, colorectal cancer, colon cancer rectal cancer, esophageal cancer, liver cancer and pancreatic cancer.

### Claim 6 (depends on 1)

The method of claim 1, wherein the human subject is an Asian subject.

### Claim 7 (depends on 1)

The method of claim 1, wherein the nucleic acid is DNA.

### Claim 8

A method of treating gastrointestinal cancer in a human subject, the method comprising:
a) detecting and/or determining the presence of gastrointestinal cancer in a human, wherein the detecting and/or determining comprises the steps of:
contacting a fluid biological sample from the subject with an agent for detecting the presence of a mutation in one or more focal regions with high mutation rates, wherein the fluid biological sample is selected from the group consisting of blood, plasma, serum, and combinations thereof,
detecting and/or determining the presence of the mutation in one or more focal regions with high mutation rates, wherein the one or more focal regions is selected from the group consisting of
CTCF-binding sites (CBS) overlapping regions set forth in the table 1 below:
Chromosome
Start site
End site
6
50570094
50570120
8
71000992
71001012
1
209422184
209422222
2
49173770
49173816
4
182064578
182064613
X
104435106
104435140
14
70285576
70285601
6
73122084
73122123
8
70576141
70576184
13
36552821
36552860
3
164903700
164903728
or portions or 1 to 4 bp sequences flanking the 5′ end thereof,
and non-CBS regions set forth in the table 2 below:
Chromosome
Start site
End site
7
68391104
68391132
7
136495924
136495948
2
57627616
57627640
16
8381278
8381302
5
23824204
23824224
7
67614923
67614943
8
65161396
65161420
7
4937707
4937736
12
126996666
126996686
4
5415060
5415082
X
137405623
137405655
3
171164993
171165017
4
144748744
144748764
9
25481736
25481758
2
77150455
77150477
3
104801455
104801477
X
125548690
125548710
14
83046706
83046744
or portions or 1 to 4 bp sequences flanking the 5′ end thereof,
wherein detecting and/or determining the presence of mutation in the CBS overlapping region(s), or portions or 1 to 4 bp sequences flanking the 5′ end thereof, comprises contacting the biological sample with a primer having a sequence selected from the group consisting of: SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. SEQ ID NO. 83, SEQ ID NO. 84 and combinations thereof and/or wherein detecting and/or determining the presence of mutation in the one or more focal regions comprises contacting the biological sample with a primer having a sequence selected from the group consisting of: SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 71, SEQ ID NO. 72, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, and combinations thereof,
wherein presence of mutation in the one or more focal regions detects and/or determines the presence of gastrointestinal cancer in the subject,
and
b) treating the subject detected and/or determined to have gastrointestinal cancer with a therapeutic agent for treating gastrointestinal cancer.

### Claim 9 (depends on 8)

The method of claim 8, further comprising determining in the fluid biological sample of the subject, whether mutation is present in at least one of the genes selected from the group consisting of: KRAS, TP53, RHOA, ARID1A, PIK3CA, ERBB3 and PTEN, wherein when the biological sample shows a presence of mutation in one or more focal regions and/or at least one of the genes, the subject is administered a therapeutic agent for treating gastrointestinal cancer.

### Claim 10 (depends on 8)

The method of claim 8, wherein the therapeutic agent is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy and combinations thereof.