Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer

RESEARCH ARTICLE Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer Fumiaki Watanabe1, Koichi Suzuki ID1*, Sawako Tamaki1, Iku Abe1, Yuhei Endo1, Yuji Takayama1, Hideki Ishikawa1, Nao Kakizawa1, Masaaki Saito1, Kazushige Futsuhara1, Hiroshi Noda1, Fumio Konishi2, Toshiki Rikiyama1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Surgery, Saitama Medical Center, Jichi Medical University, Amanuma-cho, Omiya-ku, Saitama, Japan, 2 Nerima Hikarigaoka Hospital, Hikarigaoka, Nerima-ku, Tokyo, Japan * Abstract OPEN ACCESS Citation: Watanabe F, Suzuki K, Tamaki S, Abe I, Endo Y, Takayama Y, et al. (2019) Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer. PLoS ONE 14(12): e0227366. https://doi. org/10.1371/journal.pone.0227366 Editor: Surinder K. Batra, University of Nebraska Medical Center, UNITED STATES Received: June 23, 2019 Accepted: December 17, 2019 Published: December 31, 2019 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0227366 Copyright: © 2019 Watanabe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Background Liquid biopsies enable the detection of circulating tumor DNA (ctDNA). However, the clinical significance of KRAS-mutated ctDNA for pancreatic cancer has been inconsistent with respect to its prognostic and predictive potential. Methods and findings A total of 422 blood samples were collected from 78 patients undergoing treatments for localized and metastatic pancreatic ductal adenocarcinoma. KRAS mutation in tissues and KRAS ctDNA levels in plasma were determined by RASKET and droplet digital polymerase chain reaction. Longitudinal monitoring of KRAS ctDNA was performed to assess its significance for predicting recurrence and prognosis and for evaluating therapeutic responses to chemotherapy compared with carbohydrate antigen 19–9 (CA19-9). In 67 tumor tissues, discrepancies in point mutations of KRAS were rarely observed among individual patients, implying that one targeted point mutation of KRAS can be determined in tumor tissues prior to longitudinal blood monitoring. One-time blood assessment of KRAS-mutated ctDNA before surgery or chemotherapy was not clearly associated with recurrence and prognosis. Sequential blood monitoring was performed in 39 patients who underwent surgery for potentially resectable tumors. Increased CA19-9 levels were significantly associated with recurrence, but not prognosis (P<0.001, P = 1.0, respectively), whereas emergence of KRAS ctDNA was significantly associated with prognosis (P<0.001) regardless of recurrence. Furthermore, in 39 patients who did not undergo surgery, detection of KRAS ctDNA was a predictive factor for prognosis (P = 0.005). Multivariate analysis revealed that detection of KRAS ctDNA was the only independent prognostic factor regardless of tumor resection (hazard ratios = 54.5 for patients who underwent surgery and 10.1 for patients who did not undergo surgery; P<0.001 for both). Patients without emergence of KRAS ctDNA within 1 PLOS ONE | https://doi.org/10.1371/journal.pone.0227366 December 31, 2019 1 / 17 Longitudinal monitoring of ctDNA in pancreatic cancer Funding: The present study was supported by Grant-in-Aid for Scientific Research (grant number JP 16K10514) from the Ministry of Education, Culture, Sports, Science and Technology and the JKA Foundation through its promotion funds from the Keirin Race (grant number 27-1-068 (2)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: AJCC, American Joint Committee on Cancer; CA19-9, carbohydrate antigen 19–9; ctDNA, circulating tumor DNA; ddPCR, droplet digital polymerase chain reaction; FFPE, formalinfixed paraffin-embedded; KRAS, Kirsten rat sarcoma viral oncogene homolog; OS, overall survival; PDAC, pancreatic ductal adenocarcinoma; PFS, progression-free survival; RECIST, Response Evaluation Criteria in Solid Tumors; RFS, recurrence-free survival. year after surgery showed significantly better prognosis irrespective of recurrence (P<0.001). No detection or disappearance of KRAS ctDNA within 6 months of treatment was significantly correlated with therapeutic responses to first-line chemotherapy (P<0.001). Changes in KRAS status provided critical information for the prediction of therapeutic responses. Conclusions Our study showed for the first time that detection of KRAS ctDNA levels within a short period enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer. Introduction Pancreatic ductal adenocarcinoma (PDAC) ranks as the fourth leading cause of cancer-related mortality in the United States and Japan [1, 2]. Surgical resection is considered the only curative treatment for PDAC. As PDAC is usually diagnosed at advanced stage, up to 20% of patients are suitable for initial resection [3]. Even after curative resection, most patients will experience recurrence within a year. The 5-year survival rate for patients undergoing complete resection is only approximately 25% [4]. Neoadjuvant chemotherapy to improve the treatment efficacy of surgery for patients with resectable tumors is under trial. The probability of complete resection may be lost even with a short period of neoadjuvant chemotherapy. Treatment without surgery results in unsatisfactory outcomes and poor prognosis with a median survival of 5–9 months [4], as observed in patients with unresectable tumors; nevertheless, unnecessary surgical treatment should be avoided. Recent improvements in chemotherapy for patients with unresectable tumors have shown to prolong survival. The most effective treatment should be determined according to survival benefit for each patient; hence, an ideal predictive biomarker is required. For this purpose, carbohydrate antigen 19–9 (CA19-9) has been commonly used to establish diagnosis, assess resectability, monitor progression, and determine prognosis [5]. Despite the acceptance of the utility of CA19-9 as a valuable predictor for prognosis of PDAC, controversy remains as to the sensitivity and cutoff value of CA19-9. As an alternative to (...truncated)