Telomeres are repeated DNA sequences that cap the ends of each chromosome. Telomeres are temporally among the earliest genomic sequences to be degraded during apoptosis. We quantified telomeres in cell-free DNA (CF-DNA) in vitro and in vivo. CF-DNA was obtained by differential centrifugation of media or serum to remove intact cells and cellular debris. Telomere sequence in CF-DNA was measured with a quantitative PCR-based assay. We initially studied human breast cancer and brain cancer cell lines in vitro. We found that following treatment with doxorubicin, telomere sequences were rapidly detected in CF-DNA in the media (within 24-48 hours). We call these CF-DNA telomere sequences “extracellular telomeres” (ETs). ETs were preferentially secreted after chemotherapy. The telomere sequence molar fraction in the CF-DNA was up to 50,000-fold higher compared with other genomic sequences such as exons or structural DNA sequences such as alpha-satellite DNA. CF-DNA from the serum of 80 patients with a history of breast cancer and 40 normal female volunteers were analyzed. The patients with a history of metastatic breast cancer (n = 40) had 8-fold higher median value for ETs compared to those with a history of localized breast cancer (p=0.006). 20 normal women had ET levels similar to the local breast cancer patients. We thus hypothesized that the serum extracellular telomere assay could be used as a surrogate marker for in vivo apoptotic cell death.
We collected sequential peripheral blood samples of newly diagnosed AML patients with a minimum peripheral circulating blast count of 2500/mm3 or more who received standard induction chemotherapy (anthracycline and cytarabine). These sequential peripheral blood samples were collected before, during and after standard chemotherapy at 12 hours interval for consecutive 21 days. The samples were immediately centrifuged to isolate CF DNA. Subsequently, ETs were quantitatively measured by qPCR telomere assay. In our preliminary data analysis of seven patients, we routinely observed two peaks of ETs about 10- fold higher than baseline that occurred about 2-3 and 4-6 days after initiating standard chemotherapy respectively. We conclude that 1) Telomere DNA is preferentially released from cancer cells in response to chemotherapy-induced apoptosis, 2) ETs can be detected and measured in normal patients and patients with a history of cancer, and 3) ETs are released after chemotherapy in AML patients. We are further investigating the relationship between ETs and other prognostic features in AML including initial white blood cell count, cytogenetics, disease free survival and overall survival.
Citation Format: Amitkumar Mehta, Mallick Hossain, Christine Pressey, Johanna Tuomela, Varun Dhulipala, Sunil Rangarajan, Robert Crescentini, Arja Jukkola-Vuorinen, Katri Selander, David Graves, Kevin Harris, Uma Borate. Extracellular telomeres can be detected in serum following cellular apoptosis in vitro and after chemotherapy in acute myelogenous leukemia (AML) patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1761. doi:10.1158/1538-7445.AM2013-1761