Abstract 541: Application of novel RNA aptamer-based RNase I activity assay for pancreatic cancer biomarker development
Authors: Urayama Shiro , Yu Aiming M. 
Published: August 2015

Background: Ribonuclease I (RNase I) is highly expressed in pancreas and also seen in serum. Previous reports have noted its differential activity and association to a number of cancers including pancreatic adenocarcinoma (PDAC). In search of clinically relevant biomarkers for PDAC detection, in this study, we have pursued classification characterization by a novel plasma RNase I activity assay for discriminating PDAC cases from benign conditions and other malignancies.

Methods: We have developed a new assay for accurate and precise quantification of RNase activities based on a specific and strong fluorescence intensity of malachite green (MG) bound to an RNA aptamer (MGA). The MGA chimeras were produced in E. coli using a novel optional noncoding RNA scaffold (OnRS) based recombinant RNA technology established in our lab. MGA chimeras were thus purified to a high degree of homogeneity (&gt;98% pure) with an anion-exchange fast protein liquid chromatograph (FPLC) method and utilized in our assays. The cleavage of MGA by RNases (dominantly by RNase I) results in a dose- and time-dependent decrease in the fluorescence intensity at 630/650 nm (excitation/emission) for MGA-bound MG, which could be abolished by RNase inhibitor and utilized to determine RNase activities in any given sample. We applied this assay to quantitate the serum RNase activity levels in a set of prospectively collected serum from our IRB-approved Pancreas Registry including PDAC, benign cases (including chronic pancreatitis) and other (non-PDAC) malignancies, and normalized to that of a pooled serum sample from healthy subjects. Data analysis was performed for group comparison of the RNase activity levels and significance evaluated by Mann-Whitney U test (P&lt;0.05 significant).

Results: A total of 140 patients’ serum specimen was analyzed. 60 PDAC cases (stage I (n = 8), stage II (n = 25), stage III/IV (n = 27)), 60 benign conditions (chronic pancreatitis (n = 15), acute pancreatitis (n = 3), benign cysts (n = 17), normal pancreas (n = 25)), and 20 non-PDAC malignancies (Pancreatic neuroendocrine tumor (n = 11), CholangioCA(n = 2), Ampullary CA(n = 3), Hepatoma (n = 2), GIST (n = 1), lymphoma (n = 1))

Group comparison of RNase activity:

PDAC vs benign: Median PDAC = 1.454; median benign group = 1.181 (P-value, &lt;0.0001) Resectable-stage PDAC (stages I & II) vs benign: Median PDAC = 1.616; median benign group = 1.181 (P-value, &lt;0.0001)

PDAC vs other Malignancies: Median PDAC = 1.454; median other Malignancies = 1.242 (P-value = 0.0748)

Conclusion: RNase activity level successfully discriminates PDAC cases from the benign conditions including chronic pancreatitis. Specifically, early-stage PDAC group is significantly separated from the benign group. Further testing is being performed for evaluating the test as a potential early detection diagnostic modality.

Citation Format: Shiro Urayama, Aiming M. Yu. Application of novel RNA aptamer-based RNase I activity assay for pancreatic cancer biomarker development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2015-541