Telomere length (TL) is a recognized biomarker in cancer. Epidemiology studies suggest that blood and/or buccal cell TL is associated with certain cancers; however, TL measurement is still an under-utilized biomarker in clinical trials. The molecular testing platforms currently used for TL determination are not optimal due to inherent technical limitations. While qPCR based assays have high throughput and are inexpensive, they are difficult to develop and are very sensitive to subtle permutations in reaction components. We evaluated a strategy that relies on direct hybridization and detection of the telomere target utilizing the NanoString nCounter System (NS). The NS is a semi-quantitative assay that compares the number of reporter probe hybridization events of telomere-specific versus chromosomal invariant regions in DNA samples. Performed without nucleic acid amplification, it has potential to more accurately quantify telomeric sequences than any other assay currently in use. NS uses moderate DNA quantities per test (600ng), and has a throughput that can be expanded in multiples of 12. In each sample, the Ratio of Telomere Length (RTL) is obtained by normalizing the signal produced by the telomere specific code set probes to the signal produced by multiple control probes as compared to commercial normal human DNA.

NS was validated in a three stage process using commercial DNA (n=3), cell lines (n=11) and human clinical samples (n=43) that had previously been tested by other TL tests. A blinded panel of six research samples previously tested in-house by multiplex monochrome quantitative PCR (qPCR) was sent to NanoString for in-house testing. NS exactly reproduced the panel's previous RTL ranking. The telomere code sets were subsequently tested at NCI on a panel of 11 established cell lines. Good correlation was observed compared with our in-house qPCR assay (R2 = 0.89). For the third stage of the validation, we evaluated the assay's utility on human clinical samples for which 43 whole blood leukocyte DNA samples were tested (18 dyskeratosis congenita (DC) patients with very short TL, 25 mutation-free (normal) DC relatives) and compared against leukocyte flow cytometry with in situ hybridization (flow-FISH, FF) TL measurement; FF is the gold standard in clinical testing for DC. The correlation between NS and FF lymphocyte TL was high, R2 = 0.72. Notably, the two sample t test showed that the NS assay could discriminate RTL between normals and DC patients (p = 4.6x10-8); normal relatives have longer telomeres. These data suggest that the NS RTL assay has the ability to be specific and sensitive to differences in TL between individuals with DC and healthy individuals. To determine the assay's exact clinical parameters a greater number of samples will need to be tested. The NS assay is less costly than the FF test by at least one half and if shown to be reproducible would be a viable alternative to FF and qPCR, especially in a diagnostic setting.

Citation Format: Daniel C. Edelman, David Petersen, Allison Gomez, Shahinaz Gadalla, Philippa Webster, Lucas Dennis, Mike Krouse, Sharon Savage, Paul S. Meltzer. Development of a direct-measurement molecular assay to determine telomere length in human samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5100. doi:10.1158/1538-7445.AM2014-5100