# US 11,898,208 B2 · Diagnostic method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,898,208 B2 - Document ID: US11898208B2 - Status: Granted - Kind: B2 - Application number: 16/555,609 - Filed: 2019-08-29 - Published: 2020-03-05 - Granted: 2024-02-13 - Earliest claimed: 2006-09-27 - Assignees: The Chinese University of Hong Kong - Applicants: The Chinese University of Hong Kong - Inventors: Yuk-Ming Dennis Lo; Kwan Chee Allen Chan; Chunming Ding - Web page: https://patentgrep.com/patent/US11898208B2 ## CPC - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C12Q 2600/118: Prognosis of disease development - C12Q 2600/154: Methylation 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 The present invention concerns a method for the detection or monitoring of cancer using a biological sample selected from blood, plasma, serum, saliva, urine from an individual, said method comprising: (a) obtaining DNA from the said biological sample; (b) digesting the DNA sample with one or more methylation-sensitive restriction enzymes; (c) quantifying or detecting a DNA sequence of interest after step (b), wherein the target sequence of interest contains at least two methylation-sensitive restriction enzyme recognition sites; and (d) comparing the level of the DNA sequence from the individual to a normal standard, to detect, prognosticate or monitor cancer. ## Claims ### Claim 1 A method for prognostication of hepatocellular carcinoma survival using a biological sample from an individual suffering from hepatocellular carcinoma, the method comprising: (a) obtaining DNA from the biological sample, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, and urine; (b) digesting the DNA with one or more methylation-sensitive restriction enzymes, wherein the one or more methylation-sensitive restriction enzymes preferentially cleave DNA sequences when present in an unmethylated state than in a methylated state; (c) detecting a target sequence in the DNA digested in (b), wherein the target sequence comprises at least one segment of RASSF1A promoter sequence comprising at least two methylation-sensitive restriction enzyme recognition sites; and (d) prognosticating hepatocellular carcinoma survival based on the level of the target sequence determined in (c). ### Claim 2 (depends on 1) The method according to claim 1, further comprising quantifying a trend of the level of the target sequence over a time course of one month of treatment, monitoring, or post-treatment of the hepatocellular carcinoma. ### Claim 3 (depends on 1) The method according to claim 1, wherein the target sequence further comprises exon 1 of RASSF1A. ### Claim 4 (depends on 1) The method according to claim 1, wherein following (b), each DNA from the biological sample is cleaved by at least one methylation-sensitive restriction enzyme when present in an unmethylated state. ### Claim 5 (depends on 1) The method according to claim 1, wherein in (b) the DNA is treated under conditions sufficient to allow digestion of the DNA by (i) increasing a quantity of methylation-sensitive restriction enzyme in a composition comprising the DNA, or (ii) extending an incubation time of a composition comprising the DNA. ### Claim 6 (depends on 1) The method according to claim 1, wherein (c) comprises using real-time quantitative polymerase chain reaction (Q-PCR) to generate a PCR amplicon, and wherein the PCR amplicon comprises at least two recognition sites for the one or more methylation-sensitive restriction enzymes used in (b). ### Claim 7 (depends on 1) The method according to claim 1, wherein (c) comprises using polymerase chain reaction (PCR) to generate a PCR amplicon, and wherein the PCR amplicon comprises at least two recognition sites for the one or more methylation-sensitive restriction enzymes used in (b). ### Claim 8 (depends on 1) The method according to claim 1, wherein the target sequence in (c) further comprises at least a portion of a second gene which demonstrates an aberrant DNA methylation pattern in cancer. ### Claim 9 (depends on 8) The method according to claim 8, wherein the second gene is selected from the group consisting of APC, DAP-kinase, E-cadherin, GSTPI, hMLH1, MGMT, NORE1A, p14, p15, P16INK4a, RARbeta, SOCS1, Rb, and VHL. ### Claim 10 (depends on 1) The method according to claim 1, wherein the target sequence comprises residues 1142 to 1269 of SEQ ID NO: 1. ### Claim 11 (depends on 10) The method according to claim 10, further comprising amplifying the target sequence using a primer comprising SEQ ID NO: 2 and a primer comprising SEQ ID NO: 3; and/or detecting the target sequence using a detectably-labelled probe comprising SEQ ID NO: 4. ### Claim 12 (depends on 1) The method according to claim 1, further comprising quantifying or detecting a control DNA sequence from the biological sample that has been digested with the one or more methylation-sensitive restriction enzymes, wherein the control DNA sequence does not demonstrate an aberrant DNA methylation pattern in cancer. ### Claim 13 (depends on 12) The method according to claim 12, wherein the control DNA sequence and the target sequence have a same number of methylation-sensitive restriction enzyme recognition sites. ### Claim 14 (depends on 12) The method according to claim 12, wherein the control DNA sequence comprises at least two methylation-sensitive restriction enzyme recognition sites. ### Claim 15 (depends on 1) The method according to claim 1, further comprising quantifying a trend of the level of the target sequence over a time course of one month of treatment, monitoring, or post-treatment.