Aberrant reduction of telomere repetitive sequences in plasma cell-free DNA for early breast cancer detection.

Xi Wu,Hiromi Tanaka

doi:10.18632/oncotarget.5083

Abstract

Excessive telomere shortening is observed in breast cancer lesions when compared to adjacent non-cancerous tissues, suggesting that telomere length may represent a key biomarker for early cancer detection. Because tumor-derived, cell-free DNA (cfDNA) is often released from cancer cells and circulates in the bloodstream, we hypothesized that breast cancer development is associated with changes in the amount of telomeric cfDNA that can be detected in the plasma. To test this hypothesis, we devised a novel, highly sensitive and specific quantitative PCR (qPCR) assay, termed telomeric cfDNA qPCR, to quantify plasma telomeric cfDNA levels. Indeed, the internal reference primers of our design correctly reflected input cfDNA amount (R(2) = 0.910, P = 7.82 × 10(-52)), implying accuracy of this assay. We found that plasma telomeric cfDNA levels decreased with age in healthy individuals (n = 42, R(2) = 0.094, P = 0.048), suggesting that cfDNA is likely derived from somatic cells in which telomere length shortens with increasing age. Our results also showed a significant decrease in telomeric cfDNA level from breast cancer patients with no prior treatment (n = 47), compared to control individuals (n = 42) (P = 4.06 × 10(-8)). The sensitivity and specificity for the telomeric cfDNA qPCR assay was 91.49% and 76.19%, respectively. Furthermore, the telomeric cfDNA level distinguished even the Ductal Carcinoma In Situ (DCIS) group (n = 7) from the healthy group (n = 42) (P = 1.51 × 10(-3)). Taken together, decreasing plasma telomeric cfDNA levels could be an informative genetic biomarker for early breast cancer detection.

Highlights

Telomeres are protective DNA structures that are located at the end of chromosomes, and proper telomere maintenance is indispensable for chromosomal integrity and overall genomic stability
After extracting cell-free DNA (cfDNA), we carefully measured the cfDNA concentration by a picogreen binding assay to ensure that each cfDNA concentration was at least 10 pg/μL, because this concentration provides the minimum amount of input to maintain the accuracy of the quantitative PCR (qPCR) assay and produce consistent results
Our present study focused on establishing a new strategy to assess circulating cfDNA for detecting early breast cancer

Summary

Introduction

Telomeres are protective DNA structures that are located at the end of chromosomes, and proper telomere maintenance is indispensable for chromosomal integrity and overall genomic stability. Telomere maintenance is normally controlled by telomerase activity as well as telomerase-associated factors throughout the cell cycle in tissue- and cell type-specific manners [1,2,3]. When telomere maintenance is disrupted by excessive erosion of telomeric DNA or loss of telomere binding protein function, the cellular DNA damage response (DDR) becomes activated to repair the dysfunctional telomere [5]. DDR induces a permanent proliferation arrest known as replicative senescence, which is thought to function as a tumor suppressor [6,7,8]. Dysregulation of the DDR pathway allows cells to proliferate beyond senescence limits. When the cells reach a stage of persistent telomere dysfunction (termed telomere crisis), these aberrant processes lead to telomere fusions causing genomic instability via breakage-fusion-bridge (BFB) cycles. Telomere crisis, characterized by extensive telomere erosion, chromosomal fusion, and genomic rearrangements, is an important early event in cancer development [9,10,11]

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