Abstract 4917: Identification of genetic heterogeneity by whole exome sequencing using cell free DNA from blood samples
Authors: Asada Kayo , Oda Katsutoshi , Gotoh Kengo , Kurikawa Reiko , Yamamoto Shogo , Tatsuno Kenji , Ueda Hiroki , Ikeda Yuji , Uehara Yuriko , Sone Kenbun , Hiraike-Wada Osamu , Kawana Kei , Yano Tetsu , Osuga Yutaka , Fujii Tomoyuki , Aburatani Hiroyuki 
Published: August 2015

Abstract
Objectives: Circulating cell-free DNA (cfDNA) is expected to be useful for diagnosis of genetic alterations in cancer. However, it has been still difficult to comprehensively analyze somatic mutations using cfDNA, because of the low quantity in blood. We examined the feasibility of whole exome sequencing using cfDNA and assessed the ratio of mutation reads in cfDNA for somatic mutations comparing with the corresponding tumor DNA in ovarian cancer specimens. In addition, we assessed whether cell free DNA might reflect the tumor heterogeneity in ovarian cancer.
Methods: The pair of fresh frozen tumor samples and blood samples was obtained from two cases of ovarian serous adenocarcinoma under informed consent and approval of institutional review board. The tumor DNA and cfDNA were extracted from the specimens and whole-exome sequencing was performed using 1ug of the tumor DNA or 25ng of cfDNA. The list of the mutated genes was compared between cell free DNA and the corresponding tumor DNA. Validation of the mutations and calculation of the tumor content ratio in cfDNA was assessed using target sequencing.
Result: We successfully detected the somatic mutations from cfDNA. The somatic mutations detected in cfDNA covered from 21% (49/233) to 55% (141/258) of the mutations detected in the corresponding tumor DNA. TP53 was included in the mutated genes, and the base substitution (chr17:7578257 C&gt;A) was identified in 5 of 121 reads (4%) in cfDNA. We validated the tumor content ratio of cfDNA is 8% by target sequencing. One of the two cases showed the correlation between the mutant allele frequency in the tumor DNA and the detection ratio of the mutations in cfDNA (p = 0.004, logistic regression analysis). However, the correlation was not observed in another case (p = 0.3923, logistic regression analysis). Of note, we detected various types of mutations in cfDNA alone. One of such genes is RAPGEF2. The base substitution (ch4:160253031 C&gt;T) was detected only in cfDNA (11/53, 21%), but not in tumor DNA (0/120, 0%).
Conclusion: Our data suggest that whole exome sequencing of cfDNA is feasible. The mutations detected only in cfDNA might reflect the tumor heterogeneity, and cfDNA might provide us novel biomarkers for diagnosis of recurrence and/or metastasis. Further study is warranted to elucidate the method to utilize the genetic information from cfDNA.
Note: This abstract was not presented at the meeting.
Citation Format: Kayo Asada, Katsutoshi Oda, Kengo Gotoh, Reiko Kurikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Yuji Ikeda, Yuriko Uehara, Kenbun Sone, Osamu Hiraike-Wada, Kei Kawana, Tetsu Yano, Yutaka Osuga, Tomoyuki Fujii, Hiroyuki Aburatani. Identification of genetic heterogeneity by whole exome sequencing using cell free DNA from blood samples. [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 4917. doi:10.1158/1538-7445.AM2015-4917