Abstract 2425: Nucleosomal DNA assay development and utility as PoP biomarker.
Authors: Shi Xiaoqing (Sarah) , Bao Haifeng , Wu Yuling , Chen Xiaoru , Gold David L. , LaVallee Theresa M. , Burke Patricia A. 
Published: August 2014

Abstract
Elevated baseline levels of circulating cell-free nucleosomal DNA (nDNA) are observed in some cancer patients and may be associated with clinical outcome and tumor burden. Transient increases in nDNA in response to effective chemotherapy have been observed, indicating nDNA could be a biomarker for apoptosis and could serve as a minimally invasive proof of principal (PoP) biomarker for cancer treatments. The Cell Death Detection ELISA has been used to measure nDNA levels, but the non-quantitative nature of this assay greatly limits its application in clinical studies. Our objectives were to develop a qualified assay for semi-quantitative measurement of nDNA levels in patient plasma samples and utilize the assay in Phase 1 clinical trials to assess its utility. Here we report on nDNA assay development and its application in 2 clinical trials in hematological malignancies. In order to develop a more quantitative assay, we made nDNA standards from healthy-donor blood samples and established their stability using lyophilized plasma. Recovery of freeze-thawed nDNA standard and plasma samples from normal donors and cancer patients were performed. Methods were established for the collection, storage, and analysis of plasma samples. Samples from subjects in Phase 1 dose-escalation studies undergoing treatment with MEDI-551, an anti-CD19 ADCC-enhanced monoclonal antibody (25 subjects, 0.5 - 12 mg/Kg) or moxetumomab pasudotox (CAT-8015), an anti-CD22 immunotoxin (23 subjects, 20 - 60 μg/Kg) were collected at various time points to assess changes in nDNA with treatment. Predose samples were used to assess baseline levels of nDNA for normalizing responses, comparing differences by type of malignancy, and for prognostic purposes. We developed a semi-quantitative, highly reproducible assay with low intra- and inter-plate variability. Although initial sample thaws significantly decreased signal recovery, subsequent thaws did not. Baseline levels of patient nDNA samples were not significantly different across tumor types (DLBCL, FL, CLL, MCL, MM; P=0.1170). The time course of nDNA changes in the first cycle of treatment illustrated potential differences in mechanism of action and dosing for the 2 studies, with short pronounced fold change (FC) increases occurring early in the cycle (Day 2) for MEDI-551 (weekly dosing, cycle 1) compared to prolonged FC increases at Day 5 for CAT-8015 (dosed day 1, 3, 5 every 28 days). A trend of increased nDNA with dose (MEDI-551, P=0.1522; CAT-8015, P=0.2087) and significantly increased FC for end of treatment samples was noted for both studies (MEDI-551, P=0.0438; CAT-8015, P=0.0014). These results indicate that FC increases of nDNA in response to treatment could potentially provide a proof of principle (PoP) biomarker in some clinical oncology studies. In conclusion, we have developed a new semi-quantitative qualified assay for assessing nDNA in blood which has been utilized to support clinical drug development.
Citation Format: Xiaoqing (Sarah) Shi, Haifeng Bao, Yuling Wu, Xiaoru Chen, David L. Gold, Theresa M. LaVallee, Patricia A. Burke. Nucleosomal DNA assay development and utility as PoP biomarker. [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 2425. doi:10.1158/1538-7445.AM2013-2425