Detection of alternative lengthening of telomeres by telomere quantitative PCR

Abstract

Alternative lengthening of telomeres (ALT) is one of the two known telomere length maintenance mechanisms that are essential for the unlimited proliferation potential of cancer cells. Existing methods for detecting ALT in tumors require substantial amounts of tumor material and are labor intensive, making it difficult to study prevalence and prognostic significance of ALT in large tumor cohorts. Here, we present a novel strategy utilizing telomere quantitative PCR to diagnose ALT. The protocol is more rapid than conventional methods and scrutinizes two distinct characteristics of ALT cells concurrently: long telomeres and the presence of C-circles (partially double-stranded circles of telomeric C-strand DNA). Requiring only 30 ng of genomic DNA, this protocol will facilitate large-scale studies of ALT in tumors and can be readily adopted by clinical laboratories.

ALTernative Telomere Maintenance and Cancer

“…Developed to specifically amplify C-circles, a partially single-stranded species of ECTR, the CC assay was shown to be specific and responsive to changes in ALT activity, and detected C-circles in the blood of ALT-positive osteosarcoma patients. When combined with quantitative PCR (qPCR) methods and telomere length, the CC assay may allow for rapid determination of ALT status of tumors, with high sensitivity and specificity [66]. The rapidity and ease of these tests make them well suited for clinical application.…”

Assembly of telomeric chromatin to create ALTernative endings

“…Some of the hallmarks of ALT, such as ALT associated PML bodies (APBs) and extra-chromosomal telomeric repeats (ECTR) (Box 1), have previously been observed in cells that do not display true ALT activity, when thoroughly tested [33,34]. We suggest that a more detailed analysis of several ALT hallmarks [35], for instance, using other ALT diagnostics such as the polymerase chain reaction (PCR)-based C-circle assay that detects extra-chromosomal telomeric DNA [36], might be warranted to strengthen the case for ALT activity in such tumors.…”

Alternative lengthening of telomeres: remodeling the telomere architecture

“…In addition, through its interaction with the helicases BLM and WRN, TRF2 is also involved in the unwinding of duplex telomeric DNA (Opresko et al, 2002) and potentially in the resolution of aberrant telomeric structures. The total level of TRF2 in ALT cells is not significantly different from other cells (Lovejoy et al, 2012), but the total quantity of telomeric DNA is significantly increased (Lau et al, 2012), and overexpression of TRF2 is able to suppress the formation of TIFs (Cesare et al, 2009). These observations suggest that the amount of TRF2 (and possibly of other shelterin components) relative to telomeric DNA is decreased in ALT cells, resulting in a partial functional deficiency that may contribute to the prevalence of intermediate-state TIFs in these cells, and an HR-permissive telomeric state.…”