Exploring the Alternative Lengthening of Telomeres through in vitro gene editing and the study of soft tissue sarcomas

The majority of cancers reactivate telomerase to maintain their telomere length but a minority (10 to 15%) utilize an alternative lengthening of telomeres (ALT) pathway, which involves a specialized form of homologous recombination. The CTC1/STN1/TEN1 (CST) complex plays multiple roles in telomerase-expressing cells but its role in ALT remains poorly studied. As there is more than one mechanism by which telomeres can be lengthened in ALT, the requirement for CST was investigated in three separate ALT cell lines. Using CRISPR/Cas9 to target STN1, knock-out clones were generated: loss of STN1 had little impact on cellular proliferation and had an effect only on one ALT-associated marker, C-circles, but in a cell line-dependent manner.

Soft tissue sarcomas (STSs) display a higher frequency of ALT than most other cancers; as part of our sarcoma study, DNA from non-metastatic STSs was analysed. The proportion of ALT positive tumours in our cohort was in keeping with that observed in previous studies. Little correlation between ALT status and clinical characteristics was found. Looking for signs of genomic instability, evidence of telomere fusions was found in both ALT negative and ALT positive leiomyosarcomas. Drug sensitivity tests were performed by collaborators on tumour samples, with preliminary data suggesting that drugs used for the treatment of other malignancies could be of potential benefit for STS patients.

There are currently no biomarkers to monitor disease recurrence in STS patients. Small quantities of circulating tumour DNA (ctDNA) can be found in the blood of cancer patients; the potential of ctDNA as a biomarker was therefore investigated. Using targeted Next Generation Sequencing (tNGS), ctDNA was only detected in 10% of patients. Taken together with previous results from the study, this suggests that ctDNA might be of limited use as a biomarker in non-metastatic STS patients.