# US 8,999,643 B2 · Methods and assays for the detection of alternative lengthening of telomeres (ALT) activity in cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,999,643 B2 - Document ID: US8999643B2 - Status: Granted - Kind: B2 - Application number: 13/497,473 - Filed: 2010-09-21 - Published: 2012-12-27 - Granted: 2015-04-07 - Earliest claimed: 2009-09-22 - Assignees: Children's Medical Research Institute - Applicants: Roger Robert Reddel; Jeremy David Henson - Inventors: Roger Robert Reddel; Jeremy David Henson - Web page: https://patentgrep.com/patent/US8999643B2 ## CPC - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 2600/112: Disease subtyping, staging or classification - C12Q 2600/136: Screening for pharmacological compounds - C12Q 2600/158: Expression 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 invention relates to methods and assays for the detection of active Alternative Lengthening of Telomeres (ALT) activity in cells. The methods and assays involve detecting or assaying for partially double-stranded telomeric circles wherein the presence of said circles is specific for cells comprising an active ALT mechanism. In some embodiments the methods find application in, inter alia, determining the level of ALT activity in a cell, determining the ALT status of a cancer in a subject, diagnosing and/or treating disease, determining disease status, analysis of treatment efficacy, and the identification of novel therapeutic agents. ## Claims ### Claim 1 A method for determining whether a human cell possesses an active Alternative Lengthening of Telomeres (ALT) mechanism, the method comprising: (a) amplifying partially double-stranded C-rich telomeric circles from a DNA sample from a human cell using rolling circle amplification, wherein said amplification uses the partially double-stranded C-rich telomeric circles of the DNA sample as a template and wherein said amplification is performed in the absence of an exogenous primer; (b) detecting the amplified partially double-stranded C-rich telomeric circles from step (a); and (c) determining that the human cell possesses an active ALT mechanism by the presence of the amplified circles. ### Claim 2 (depends on 1) A method according to claim 1 wherein the partially double-stranded C-rich telomeric circles comprise repeats of the sequence (CCCTAA)n on a circular strand and comprise the sequence (TTAGGG)n on a linear strand. ### Claim 3 (depends on 1) A method according to claim 1 wherein a circular and/or a linear strand comprises variant telomeric repeat sequences, mutant telomeric sequences and/or further comprise non-telomeric sequences. ### Claim 4 (depends on 1) A method according to claim 1 wherein the cell is a cancer cell. ### Claim 5 (depends on 1) A method according to claim 1 wherein the cell is derived from a subject suffering from, suspected of suffering from, or predisposed to, a disease or condition associated with abnormal cellular proliferation. ### Claim 6 (depends on 5) A method according to claim 5 wherein the disease or condition is a cancer selected from a sarcoma, a blastoma, a carcinoma, a mesothelioma or an astrocytoma. ### Claim 7 (depends on 1) A method according to claim 1, wherein the method comprises: (1) optionally isolating DNA from the cell; (2) incubating the DNA in the presence of a DNA polymerase and one or more dNTPs under suitable conditions such that polymerase-mediated extension from a linear strand of the partially double-stranded telomeric DNA generates concatemers of single-stranded telomeric DNA; and (3) detecting the concatemers. ### Claim 8 (depends on 7) A method according to claim 7 wherein the concatemers are detected by hybridisation, sequencing, PCR, molecular beacons, or nucleic acid enzymes, or by incorporating suitably labelled dNTPs in incubation step (2). ### Claim 9 (depends on 7) A method according to claim 7 wherein the DNA polymerase is φ29 DNA polymerase. ### Claim 10 (depends on 1) A method according to claim 1 wherein the partially double-stranded C-rich telomeric circles are amplified from a biological sample derived from a subject. ### Claim 11 (depends on 10) A method according to claim 10 wherein the biological sample comprises blood, urine, sputum, pleural fluid, peritoneal fluid, bronchial or bronchioalveolar lavage fluid, or a tissue section. ### Claim 12 (depends on 11) A method according to claim 11 wherein the blood is whole blood, blood serum or blood plasma. ### Claim 13 A method for determining the level of Alternative Lengthening of Telomeres (ALT) activity in a human cell, the method comprising: (a) amplifying partially double-stranded C-rich telomeric circles from a DNA sample from a human cell using rolling circle amplification, wherein said amplification uses the partially double-stranded C-rich telomeric circles of the DNA sample as a template, and wherein said amplification is performed in the absence of an exogenous primer; (b) detecting the amplified partially double-stranded C-rich telomeric circles from step (a); and (c) determining the level of ALT activity in the human cell by the amount of the amplified circles, wherein the amount of the amplified circles is indicative of the level of ALT activity in the human cell. ### Claim 14 A method for determining the Alternative Lengthening of Telomeres (ALT) status of a cancer in a human subject, the method comprising: (a) obtaining a sample from the subject; (b) amplifying partially double-stranded C-rich telomeric circles from the sample using rolling circle amplification, wherein said amplification uses the partially double-stranded C-rich telomeric circles of the DNA sample as a template, and wherein said amplification is performed in the absence of an exogenous primer; (c) detecting the amplified partially double-stranded C-rich telomeric circles from step (b); and (d) determining the ALT status of a cancer in the human subject, wherein the presence of the amplified circles is indicative of the human subject having cancer cells possessing ALT activity, and wherein the absence of the amplified circles is indicative of the human subject having cancer cells not possessing ALT activity. ### Claim 15 (depends on 14) A method according to claim 14 further comprising quantifying the level of ALT activity in the cancer cell and/or quantifying the amount or proportion of ALT+ cells in the sample based on the amount of the partially double-stranded C-rich telomeric circles in the sample. ### Claim 16 A method for diagnosing cancer, or predicting the onset thereof, in a human subject, wherein the cancer displays Alternative Lengthening of Telomeres (ALT) activity, the method comprising: (a) obtaining a sample from the human subject; (b) amplifying partially double-stranded C-rich telomeric circles from the sample using rolling circle amplification, wherein said amplification uses the partially double-stranded C-rich telomeric circles of DNA as a template and wherein said amplification is performed in the absence of an exogenous primer; (c) detecting the amplified partially double-stranded C-rich telomeric circles from step (b); and (d) diagnosing cancer, or predicting the onset thereof, wherein the presence and/or amount of the amplified circles is indicative of the human subject having cancer, or the likelihood of the onset of cancer in the human subject. ### Claim 17 (depends on 16) A method according to claim 16 wherein the human subject is suspected of having cancer, is predisposed or otherwise susceptible thereto, has one or more high risk factors for developing cancer, or has a cancer predisposition syndrome. ### Claim 18 A method for evaluating the efficacy of a treatment regime in a human subject suffering from a cancer displaying Alternative Lengthening of Telomeres (ALT) activity, the method comprising: (a) treating the subject with a suitable anti-cancer treatment regime for a period sufficient to evaluate the efficacy of the regimen; (b) obtaining a sample from the subject; (c) amplifying partially double-stranded C-rich telomeric circles from the sample using rolling circle amplification, wherein said amplification uses the partially double-stranded telomeric circles of DNA as a template, and wherein said amplification is performed in the absence of an exogenous primer (d) detecting the amplified partially double-stranded C-rich telomeric circles from step (c), (e) repeating steps (b), (c) and (d) at least once over a period of time; (f) determining whether the presence and/or amount of the amplified circles changes over the period of time; and (g) evaluating the efficacy of the treatment regimen in the human subject, wherein a change in the presence and/or amount of the amplified circles is indicative of a change in disease control in the human subject and the degree of efficacy of the treatment regime.