Abstract P1-01-14: Nuclease-activated oligonucleotide probes for detection of breast cancer circulating tumor cells (CTCs): Early clinical results

Abstract

Abstract Introduction: A challenge for CTC-based diagnostic tests has been the development of methods with sufficient sensitivity to detect low levels of CTCs. Expense, accuracy and complexity have also limited clinical uptake of CTCs. To overcome these limitations we explored detecting CTCs by measuring their nuclease activity with nuclease-activated probes. We present the development of a rapid and highly-sensitive CTC detection assay based on probes that are selectively digested (activated) by target nucleases expressed in breast cancer cells. Methods: Nuclease activity in samples from women with Stage IV breast cancer and healthy donors was determined and correlated with clinical data. Patients seen at University of Iowa Clincis were eligible for this IRB-approved study. Blood samples were processed using microfilter (ScreenCell) units for CTC enrichment and converted into cell lysates that were examined by means of three different chemically-optimized oligonucleotide probes. CTC-derived nuclease activity was quantified using a fluorometer. The presence of CTCs was confirmed using established CTC detection methods (e.g. RT-PCR, immunohistostaining). Results: Sensitivity of the probe assay was 5 cancer cells in buffer solution and ~200 cancer cells in 1 mL of healthy donor blood. The final study cohort included 28 breast cancer patients and 10 healthy donors. The averaged signal intensities from patient samples were significantly higher compared to the healthy donor control group, presumably arising from CTCs in the blood. Statistical analysis further reveald short incubations in the assay (<20 min) to be optimal. From an ROC analysis we obtained AUC values of 0.8821, 0.8103 and 0.9356 for the three different probes. The oligonucleotide probe being the best predictor of disease yielded 100% sensitivity in the patient samples with a specificity of 70%. The dsDNA 20 minute probe was correlated negatively with tumors being ER+/PR+ (p=0.03). The 2'f-RNA 0 minute probe correlated significantly with HER2- tumors (p=0.04). In this smaller series other trends were also suggested. Conclusion: We describe a novel diagnostic for the detection of CTCs that could overcome limitations of CTC detection assays and could provide a robust diagnostic tool for breast cancer. Future clinical assays derived from this technology could require minimal training and infrastructure and might be developed into a point-of-care testing format. Citation Format: Giangrande PH, Kruspe S, Dickey DD, Kamboj S, Clark KC, Urak K, Burghardt E, Smith B, Thomas A, McNamara JO. Nuclease-activated oligonucleotide probes for detection of breast cancer circulating tumor cells (CTCs): Early clinical results [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-14.