Methods and assays for the detection of alternative lengthening of telomeres (ALT) activity in cells

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.

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.

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.

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.