Abstract 5164: cell-free DNA (cfDNA) fragment size as a potential quantitative biomarker for metastatic colorectal cancer (mCRC)
Authors: Kang Jun-Kyu , Kim Hwang-Phill , Lim Yoojoo , Kim Su Yeon , Kim Tae-You 
Published: June 2022

Abstract
Introduction: The fragment size of cfDNA derived from tumor cells are known to be shorter than that from normal cells for not yet precisely known mechanisms. We hypothesized that the fragment size of cfDNA can be utilized to reliably estimate tumor burden in patients with mCRC undergoing palliative chemotherapy.
Methods: cfDNA sequencing data from mCRC patients receiving first-line palliative chemotherapy at Seoul National University Hospital (Seoul, Korea) were used for this analysis. The patients were enrolled in a main study evaluating the dynamic changes of circulating tumor DNA mutational profiles during chemotherapy. Blood samples were obtained prior to chemotherapy and after every four cycles of chemotherapy until disease progression. cfDNA sequencing data from normal individuals were used as control data. cfDNA sequencing for both cancer patients and normal individuals were performed using AlphaLiquid® 100 target capture panel (IMBdx, Inc., Seoul, South Korea). AlphaLiquid® 100 is a tumor agnostic panel consist of 106 genes, including 10 gene fusion and MSI. Based on the panel sequencing data, the genetic alterations and the fragment size of cfDNA were calculated. The fragmentation ratio was defined by the ratio of the read fragment proportion in size range P1 (100 - 155 bp) and P2 (160 - 180 bp).
Results: cfDNA sequencing data from 280 plasma samples from 62 mCRC patients and 50 healthy controls were used for analysis. Compared to cfDNA from healthy controls, the cfDNA fragment sizes from mCRC patients were significantly shorter (169.585 bp vs. 173.964 bp; p &lt; 0.001). Comparing the fragment sizes by the mutational status, the fragment sizes of alleles with detected somatic mutations were significantly shorter than those with reference alleles (mean fragment size 155.853 bp in alleles with somatic mutations vs. 160.613 bp in reference alleles; p &lt; 0.001), but such difference was not observed with germline mutations (mean fragment size 160.911 bp in alleles with germline mutations vs. 159.889 bp in reference alleles; p = 0.992). Further, the clonality inferred from the variant allele frequency (VAF) of somatic mutation was negatively correlated with the size of the cfDNA fragment. The read fragment proportion in size range P1 was significantly associated with the clonality (r = 0.86, p &lt;0.001). We calculated the mean size of DNA fragment and the fragmentation ratio with each longitudinal sample. The fragmentation ratio was found to decrease with chemotherapy in responders and re-increase at the time of resistance to chemotherapy.
Conclusions: The fragmentation ratio calculated from cfDNA in mCRC patients can represent tumor burden in dynamic samples and potentially can be used as a biomarker.
Citation Format: Jun-Kyu Kang, Hwang-Phill Kim, Yoojoo Lim, Su Yeon Kim, Tae-You Kim. cell-free DNA (cfDNA) fragment size as a potential quantitative biomarker for metastatic colorectal cancer (mCRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5164.