The definitive test for ALT activity is based on its initial discovery as the non-telomerase-mediated mechanism for preventing telomere shortening4. However, it is often not practical to prove ALT activity by showing telomere length maintenance in the absence of telomerase activity. This involves passaging cells for 30-100 population doublings, which is not possible in tumor specimens or cells with limited viability due, for example, to treatment with potential ALT inhibitors. The presence of ALT activity has therefore been inferred by detecting telomere-related phenotypes that are characteristic of most ALT[+] cell lines. The stark contrast between the long heterogeneous telomere lengths of ALT[+] cells and the much shorter, homogeneous distribution of lengths in ALT[-] cells, as demonstrated by terminal restriction fragment (TRF) analysis, is currently the gold-standard assay for ALT12, 20.
The other well-established ALT marker, the ALT-associated PML body (APB)21, has been useful for determining ALT status in tumors as it can be assayed on tumor specimens archived as paraffin-embedded specimens15, 22 and is more convenient than TRF analysis. The APB assay uses fluorescence in situ hybridization and immunostaining to demonstrate ALT-associated localization of telomeric DNA to nuclear foci of PML protein. Long heterogeneous telomeres and APBs are not completely definitive for ALT activity, however, because rare ALT[+] cell lines have been found where one or both of these features are absent23, 24 and under unusual circumstances they can be present in ALT[-] cells25. In other words, it is unlikely that the current gold standards for detecting ALT are completely definitive, and likely that this has underestimated the prevalence of ALT in tumors 8.
Recently, the first specific molecule for ALT, telomeric C-Circle DNA, was discovered and this enabled the first quantitative test for ALT activity26. C-Circles are partially single-stranded telomeric (CCCTAA)n DNA circles that are present in 1000-fold greater abundance in ALT[+] cells than ALT[-] cells (telomerase[+] or normal somatic cells). Because C-Circles appear on activation of ALT and disappear within 24 hours of ALT inhibition they may well be an integral part of the ALT mechanism. The C-Circle Assay is sensitive to a few hundred ALT[+] cells and clearly distinguishes ALT[+] cell lines from ALT[-] cells. The C-Circle Assay also gave the correct ALT status for all cell lines in which the standard ALT assays are known to give false-positive and false-negative results. C-Circle levels were also elevated in the blood from patients with ALT[+] bone cancers suggesting that C-Circles could be used as a blood-based biomarker for ALT cancer activity 26.
The C-Circle assay has opened up a range of opportunities for ALT research and the clinical detection of cancers that rely on the ALT mechanism. It is the only assay for ALT that is rapidly and linearly responsive to changes in ALT activity and hence the only assay that can be used to screen for ALT inhibitors or generally measure changes in ALT activity. Identifying ALT inhibitors or gene targets for inhibiting ALT is important as ALT inhibition could provide anti-cancer therapies with minimal side-effects 12, 27. The C-Circle assay also provides a higher throughput than current methods for determining ALT status of tumors. This could allow more extensive investigation of ALT in cancer to identify the areas where the clinical detection of ALT could help patient management. Associations of ALT with various types or subtypes of cancer may also help understanding the regulation of the ALT mechanism 12, 28, 29. The C-circle assay could also be used to improve the chance of success of clinical trials by identifying a responsive subgroup. ALT[+] and ALT[-] subgroups can have different sensitivities to anti-cancer therapies that do not specifically target TMMs19, 30.
Significantly, the C-Circle assay is the only clinically applicable ALT test and C-circles are the only potential blood based biomarker for measuring ALT activity (arising from a tumor). A clinically applicable or blood-based C-Circle assay could be used for (i) a screening test to assist either early diagnosis of primary tumors in high-risk groups or early detection of recurrences, both of which could improve patient outcome (ii) monitoring the success of chemotherapy; for example, in the 50% of bone cancer cases that are ALT[+], a quantitative blood test for ALT activity could identify the non-responsive patient group in time to allow a different or more aggressive chemotherapy regime to be instituted to improve survival15, 31 (iii) a prognostic indicator; for example, in brain tumors, ALT activity is the best known prognostic indicator14 and a (clinical laboratory applicable) C-Circle assay could allow routine identification of the ALT[+] group, which is the subgroup most likely to benefit from more aggressive management15 (iv) monitoring progression of cancer to facilitate the selection of the most appropriate management (v) determination of the most appropriate anti-cancer therapy; with the potential development of telomerase and ALT targeted anti-cancer drugs it will become necessary to know which TMM is active in the cancer.
The core of the ALT assay is isothermic amplification of C-circle complementary strand and hybridization with a sequence specific 32P-labeled probe. Sensitivity of the ALT assay is very high. The linear signal increase can be detected with the range of total genomic DNA between 1 and 50 ng (9). The C- Circle assay is being used for detecting the presence of ALT and measuring changes to ALT activity after treatment (such as RNAi).
Figure 1. C-circle (CC) assay principle. Amplification products are detected by hybridization with 32P-(CCCTAA)3 probe.