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Clinical utility of the mutational landscape and fragment size of circulating tumor DNA in renal cell carcinoma

  • Uemura M. 1,
  • Yamamoto Y. 1,
  • Fujita M. 2,
  • Maejima K. 2,
  • Koh Y. 1,
  • Matsushita M. 1,
  • Nakano K. 1,
  • Hayashi Y. 1,
  • Wang C. 1,
  • Ishizuya Y. 1,
  • Kato T. 1,
  • Kawashima A. 1,
  • Ujike T. 1,
  • Nagahara A. 1,
  • Fujita K. 1,
  • Nakagawa H. 2,
  • Nonomura N. 1
1 Osaka University, Graduate School of Medicine, Dept. of Urology, Suita, Japan 2 RIKEN Center for Integrative Medical Sciences, Laboratory for Genome Sequencing Analysis, Tokyo, Japan

Publication: March 2019

Introduction & Objectives

Reliable biomarkers for renal cell carcinoma (RCC) have yet to be found. Circulating-tumor DNA (ctDNA) is an emerging resource to detect and monitor molecular characteristics of various tumors. This study aims to clarify the clinical utility of ctDNA for RCC.

Materials & Methods

Fifty-three patients histologically diagnosed as clear cell RCC were enrolled. Targeted sequencing was performed using plasma cell-free DNA (cfDNA) and tumor DNA. We applied droplet digital PCR to validate detected mutations. cfDNA fragment size was also evaluated using a microfluidics-based platform and sequencing. Proportion of cfDNA fragments was defined as the ratio of small (50-166 bp) to large (167-250 bp) cfDNA fragments. Mutant allele frequency in ctDNA was analyzed with respect to clinical course. Prognostic potential was evaluated using log-rank test.


A total of 38 mutations across 16 (30%) patients were identified from cfDNA, including mutations in TP53 (n = 6), VHL (n = 5), BAP1 (n = 5) and TSC1 (n = 4), and the median mutant allele frequency of ctDNA was 10%. We designed specific droplet digital PCR probes for 11 mutations and detected the same mutations in both cfDNA and tumor DNA. In 5 RCC patients whose plasma cfDNA and tumor DNA were sequenced, at least two mutations were detected in cancer tissue from all 5 patients, and the corresponding mutations in ctDNA were detected for two of these patients, as shown in figure below. Overall, detection of ctDNA was significantly associated with a higher proportion of short cfDNA fragments (P = 0.033), indicating RCC patients with ctDNA had shorter fragment sizes of cfDNA. Interestingly, the changes of mutant allele frequency in ctDNA concurrently correlated with clinical course in 6 RCC patients. Positive ctDNA and fragmentation of cfDNA were significantly associated with poor cancer-specific survival (P < 0.001, P = 0.011, respectively).



Our results show that mutational landscape and fragmentation of plasma cfDNA have promising prognostic potential in RCC patients. The change of mutant allele frequency of ctDNA may be an auspicious monitoring marker for RCC. Given that plasma cfDNA is easily collected from peripheral blood, these newly discovered markers can be convenient and precise tools for understanding RCC.