Abstract 5234: Tumor-unique mutation detection in cell-free DNA to monitor colorectal tumor burden using a cancer-associated gene sequencing panel
Authors: Sato Kei A. , Nishizuka Satoshi S. , Iwaya Takeshi , Kume Kohei , Otsuka Koki , Wakabayashi Go 
Published: August 2015

Abstract
Background: Cell-free DNA (cfDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of cfDNA to quantitatively monitor tumor burden based on individual mutation profiles.
Materials and Methods: DNA was extracted from a total of 236 samples from pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells of 72 individuals who underwent curative resection of colorectal tumors and from healthy individuals. With the use of a sequencing panel comprising 50 cancer-associated genes in an Ion PGM sequencer, mutations with a frequency higher than 0.1% in the tumor were designated as tumor-unique mutations and were used to trace the tumor burden by cfDNA. These experiments aimed to identify: (a) tumor-unique mutations and (b) the potential utility of tumor-unique mutations detected in cfDNA.
Results: The mean cfDNA levels of healthy individuals, endoscopically resectable tumors, and advanced cancers were 5.1, 15.9, and 27.4 ng/mL per plasma, respectively. The group average appeared to have decreased after both endoscopic and laparoscopic procedures, suggesting that the cfDNA level may have diagnostic utility. From the panel of 50 cancer-associated genes, an average of 6.7 tumor-unique mutations were found per tumor, in which TP53 and CDKN2A were mutated in more than 60% of tumors. All tumors showed mutation in either one of these genes, whereas 15 genes were not mutated in any of the tumors. Quantitative monitoring of the mutated fragments in cfDNA with Ion PGM or droplet digital PCR demonstrated that 16 out of 49 (32.7%) mutated fragments decreased the mutation frequency corresponding to the tumor burden in individual patients after operation. These results suggest that selective tumor-unique mutations in cfDNA precisely represent tumor burden in individual patients in a quantitative manner. Although the tumor-unique mutation in genes that are frequently altered may seem to have played a dominant role in diagnostics, mutations with a minor frequency in the population, such as FBXW7, also contributed to reflect tumor-unique mutational properties.
Conclusions: The 50 cancer-associated gene panel appeared sufficient in identifying individual tumor-unique mutated cfDNA makers for cancer patients. The validation process to detect tumor-unique mutations in cfDNA has yet to be well established. However, the potential clinical utility of tumor unique-mutations as a biomarker has been demonstrated, particularly for applications, such as post-operative follow-up.
Citation Format: Kei A. Sato, Satoshi S. Nishizuka, Takeshi Iwaya, Kohei Kume, Koki Otsuka, Go Wakabayashi. Tumor-unique mutation detection in cell-free DNA to monitor colorectal tumor burden using a cancer-associated gene sequencing panel. [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 5234. doi:10.1158/1538-7445.AM2015-5234