Abstract 5232: Changes in plasma circulating DNA composition during focal ablative therapy for liver cancer
Authors: Tiirikainen Maarit I. , Peplowska Karolina , Song Min-Ae , Miyazaki Kyle , Wong Linda L. , Kwee Sandi A. 
Published: August 2015

Abstract
Circulating cell-free DNA (cfDNA) may potentially serve as a diagnostic or therapeutic biomarker. We tested our established methodologies for cfDNA profiling on blood samples obtained during focal ablative treatments for liver cancer, given that such interventions may cause abrupt spillage of circulating tumor DNA (ctDNA) into the bloodstream.
Following written informed consent, serial blood samples from 13 patients with hepatocellular carcinoma (HCC) were drawn into cfDNA-optimized collection tubes before treatment, 2-3 hours post-treatment, and a few weeks after. Nine patients underwent radiofrequency ablation and 4 patients received transarterial chemoembolization. The cfDNA concentration was assessed by quantitative PCR of Alu-amplicons (Alu-101 and Alu-226) on 13 pre-treatment samples, 12 immediate post-treatment samples and 8 follow-up samples. Aberrant cfDNA methylation at genes GSTP1 and RASSF1 was assessed by pyrosequencing to investigate tumor-specific cfDNA.
Mean cfDNA levels of patients with HCC increased by an average of 3.6-fold (up to 9.3-fold) from baseline immediately after treatment (p = 0.0019). Levels measured 1-15 weeks after treatment returned to on average 87% of the baseline. Each specimen contained both high molecular weight DNA (consistent with abrupt release of DNA from disrupted cells) and smaller apoptotic cfDNA fragments that ranged in size of 160-180 bp. DNA integrity (Alu-226/Alu-101) was similar before treatment, immediately after, and in the follow-up samples. Compared to the paired leukocyte DNA methylation levels, higher methylation levels at RASSF1 and/or GSTP1 were observed in all 13 pre-treatment cfDNA samples, supporting at least partial tumor origin of the isolated cfDNA. RASSF1 methylation moderately correlated with GSTP1 methylation (r = 0.55, p&lt;0.001). Excluding one patient with exceptionally high RASSF1 and GSTP1 methylation levels, the averaged amount of RASSF1/GSTP1-methylated ctDNA correlated with the tumor size prior to the procedure (r = 0.72, p = 0.008), while no association was found between the AFP levels and tumor burden.
The characteristics of cfDNA in cancer patients may change significantly following therapeutic interventions. We have established an improved workflow for cfDNA quantification and characterization in plasma that can be applied to the peri-treatment setting and are in the process of evaluating ctDNA analysis protocols for EDTA blood as well. These preliminary observations in a limited number of specimens support the potential of quantifying ctDNA as a marker of tumor cell destruction. Preliminary observation of a correlation between aberrantly-methylated cfDNA levels and HCC tumor size suggests that pre-treatment cfDNA profiling may inform on tumor burden. Further research will be needed to validate the clinical significance of the quantitative and epigenetic changes in cfDNA samples from patients with HCC.
Citation Format: Maarit I. Tiirikainen, Karolina Peplowska, Min-Ae Song, Kyle Miyazaki, Linda L. Wong, Sandi A. Kwee. Changes in plasma circulating DNA composition during focal ablative therapy for liver cancer. [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 5232. doi:10.1158/1538-7445.AM2015-5232