# US 2014/0155279 A1 · Detection of a panel of urine DNA markers for HCC screening and disease management

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- Number: US 2014/0155279 A1
- Document ID: US20140155279A1
- Status: Published application
- Kind: A1
- Application number: 14/079,649
- Filed: 2013-11-14
- Published: 2014-06-05
- Earliest claimed: 2012-11-14
- Assignees: JBS Science Inc.
- Applicants: JBS Science Inc.
- Inventors: Wei SONG; Batbold BOLDBAATAR; Lijia XIE; Sitong CHEN
- Web page: https://patentgrep.com/patent/US20140155279A1

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 2600/154: Methylation markers
- 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

Provided herein is a method for detecting the presence or absence of a cancer in a biological sample of an individual, by determining the level of mutation and methylation of one or more genes from a group of genes comprising TP53, CTNNB1, hTERT, RASSF1A, GSTP1, p16, p15 and SFRP-1. Also provided herein is an assay to detect p53 mutations suitable for DNA isolated from biological body fluid in order to screen cancer patients. Also provided is a method for detecting the presence or absence of a liver cancer in an individual by determining the level of methylation. Also provided is a suitable method for detecting the presence or absence of a liver cancer in an individual by determining the level of methylation of the promoter of the GSTP1 gene in body fluid such as urine or blood. Also provided is a suitable method for detecting the presence or absence of a liver cancer in an individual by determining the level of methylation of the promoter of the RASSF1A gene in body fluid such as urine or blood.

## Claims

### Claim 1

A method of detecting the presence or absence of a cancer in a biological sample of a subject in need thereof, comprising:
(v) preparing DNA from the biological sample;
(vi) determining the level of mutation or methylation of one or more genes from a group of genes comprising TP53, CTNNB1, hTERT, RASSF1A, GSTP1, p16, p15 and SFRP-1 from the biological sample of the subject;
(vii) comparing the level of mutation or methylation of the one or more genes with the level of mutation or methylation of the one or more genes in one or more control samples from subjects known not to have said cancer; and
(viii) detecting the presence or absence of the cancer, with elevated mutation or methylation levels in the one or more genes of the individual as compared to the level of mutation or methylation in the one or more genes in the one or more control samples indicating the presence of the cancer, and the absence of elevated mutation or methylation levels indicating the absence of the cancer. The method claim of 1, wherein the biological sample comprises tissue or body fluid selected from the group consisting of serum, plasma, and urine.

### Claim 2 (depends on 1)

The method of claim 1 wherein the cancer is hepatocellular carcinoma (HCC) and the biological sample is tissue, serum, plasma, or urine.

### Claim 3 (depends on 2)

The method of claim 2, wherein the level of mutation is the level of TP53 249T mutation.

### Claim 4 (depends on 2)

The method of claim 2, wherein the levels of methylation is the levels of methylation of RASSF1A or GSTP1.

### Claim 5 (depends on 4)

The method of claim 4, wherein level of methylation is the level of methylation of the regulatory region of the RASSF1A gene or GSTP1 gene.

### Claim 6 (depends on 5)

The method of claim 5, wherein the regulatory region is the promoter and the first exon of the RASSF1A gene or GSTP1 gene.

### Claim 7 (depends on 6)

The method of claim 6, wherein the regulatory region is the promoter and the first exon of the RASSF1A gene, and the regulatory region is selected from the groups consisting of: (i) P1 region of the promoter of the RASSF1A gene; (ii) P2 region of the promoter of the RASSF1A gene and (iii) E1 region of the first exon of the RASSF1A gene.

### Claim 8 (depends on 6)

The method of claim 6, wherein the level of methylation is determined by methylation specific PCR (MSP) assays or a two-step short-amplicon methylation specific PCR (shMSP).

### Claim 9 (depends on 7)

The method of claim 7, wherein the regulatory region is P1 region and the methylation specific PCR (MSP) for the P1 region uses primers of the nucleotide sequences as set forth in SEQ ID NO:7 and SEQ ID NO:8 and is probed by a probe of the nucleotide sequence as set forth in SEQ ID NO:9.

### Claim 10 (depends on 7)

The method of claim 7, wherein the regulatory region is P1 region and the two-step short-amplicon methylation specific PCR (shMSP) for the P1 region uses primers of the nucleotide sequences as set forth in SEQ ID NO: 15 and SEQ ID NO:16 for the first step PCR and SEQ ID NO:17 and SEQ ID NO:18 for the second step PCR.

### Claim 11 (depends on 2)

The method of claim 2, wherein the level of mutation is the level of mutation of TP53 gene, the biological sample is urine, and the level of mutation is deemed elevated when the copies of mutated TP53 is greater than 50 copies per milliliter of urine.

### Claim 12 (depends on 2)

The method of claim 2, wherein the level of methylation is the level of methylation of mRASSF1A gene, the biological sample is urine, and the level of methylation is deemed elevated when the copies of methylated mRASSF1A is greater than 50 copies per milliliter of urine.

### Claim 13 (depends on 2)

The method of claim 2, wherein the level of methylation is the level of methylation of mGSTP1 gene, the biological sample is urine, and the level of methylation is deemed elevated when the copies of methylated mGSTP1 is greater than 1,000 copies per milliliter of urine.

### Claim 14 (depends on 2)

The method of claim 2, wherein the biological sample is urine, and the level of mutation is the level of mutation of TP53 and the levels of methylation are the levels of methylations of RASSF1A and GSTP1.

### Claim 15 (depends on 14)

The method of claim 14, wherein the subject's probability of having HCC is determined by logistic regression modeling based on three predictor variables: (1) the number of copies of mutated TP53 per milliliter of urine; (2) the number of copies of methylated RASSF1A per milliliter of urine; and (3) the number of copies of methylated mGSTP1 per milliliter of urine, wherein the categorical dependent variable of the logistic regression model is whether the subject has HCC.

### Claim 16 (depends on 15)

The method of claim 15, wherein the subject's probability of having HCC is calculated by taking the logistic regression of the three predictor variables and a fourth predictor variable, wherein the fourth variable is 1 when the concentrations of alpha-fetoprotein (AFP) in urine is above 20 ng/ml, and the fourth variable is 0 when the AFP concentration is below 20 ng/ml.

### Claim 17 (depends on 1)

The method of claim 1, wherein the level of methylation is the methylation level of CpG sites in a selected regulatory region of the one or more gene.

### Claim 18 (depends on 17)

The method of claim 17, wherein the selected regulatory region is SEQ ID NO: 29 and the level of methylation is determined by two-step short-amplicon methylation specific PCR (shMSP), wherein the shMSP uses primers of the nucleotide sequences as set forth in SEQ ID NO:19 and SEQ ID NO:20 for the first step PCR and in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 for the second step PCR reaction.

### Claim 19

A method for identifying and quantifying at least one nucleic acid sequence from a biological sample of a subject, comprising: (1) isolating the at least one nucleic acid sequence from the biological sample; (2) contacting one or more LNA clamps with the at least one nucleic acid sequence, wherein the one or more LNA clamps correspond to the wildtype allele of the at least one nucleic acid sequence and thereby prevents amplification of the wildtype sequence but not the non-wildtype sequence; (3) amplifying the at least one nucleic acid sequence; and (4) quantifying the at least one nucleic acid sequence.

### Claim 20

The method claim of 19, wherein the biological sample comprises tissue or body fluid wherein the tissue or body fluid is selected from the group consisting of serum, plasma, and urine, wherein the LNA clamp comprises a methylene bridge connecting a 2′-oxygen and 4′-carbon (locked nucleic acid (LNA) clamp) in the wildtype nucleic acid sequence.

### Claim 21

A kit for identifying and quantifying a nucleic acid sequence from a biological sample, comprising a LNA (locked nucleic acid) clamp for a wildtype nucleic acid sequence a 5′ and a 3′ primer for the wildtype nucleic acid sequence, wherein the LNA clamp sequence is modified at a ribose to prevent amplification of the wildtype nucleic acid sequence in a nucleotide amplification reaction assay.