Antibiotics Improve the Treatment Efficacy of Oxaliplatin-Based but Not Irinotecan-Based Therapy in Advanced Colorectal Cancer Patients

Background Oxaliplatin and irinotecan are generally used to treat advanced colorectal cancer (CRC) patients. Antibiotics improve the cytotoxicity of oxaliplatin but not irinotecan in a colon cancer cell line in vitro. This study retrospectively assessed whether antibiotics improve the treatment efficacy of oxaliplatin- but not irinotecan-based therapy in advanced CRC patients. Patients and Methods. The medical records of 220 advanced CRC patients who underwent oxaliplatin- or irinotecan-based therapy were retrospectively reviewed. The oxaliplatin and irinotecan groups were further divided into antibiotic-treated (group 1) and antibiotic-untreated (group 2) subgroups. Results In oxaliplatin groups 1 and 2, the response rate (RR) was 58.2% and 30.2%, while the disease control rate (DCR) was 92.5% and 64.2%, respectively; the median progression-free survival (PFS) was 10.5 months (95% confidence interval (CI) = 7.5–12.2) and 7.0 months (95% CI = 17.0–26.0), respectively, and the median overall survival (OS) was 23.8 months (95% CI = 5.1–9.1) and 17.4 months (95% CI = 13.1–24.9), respectively. In irinotecan groups 1 and 2, the RR was 17.8% and 20.0%, while the DCR was 75.6% and 69.1%, respectively; the median PFS was 8.2 months (95% CI = 6.2–12.7) and 7.9 months (95% CI = 12.0–23.0), respectively, and the median OS was 16.8 months (95% CI = 5.9–10.6) and 13.1 months (95% CI = 10.4–23.7), respectively. Conclusion To improve the treatment efficacy of oxaliplatin-based therapy in advanced CRC patients, adding antibiotics is a potential therapeutic option.


Introduction
Oxaliplatin and irinotecan are anticancer agents used to treat cancer patients [1,2] and in colorectal cancer (CRC) patients, oxaliplatin-, and irinotecan-based therapy is generally used as first-or second-line treatment [3][4][5][6]. However, their treatment efficacy in advanced CRC patients is limited. Some types of bacteria mediate the resistance to gemcitabine in the pancreatic cancer cell line and to oxaliplatin but not to irinotecan in the colon cancer cell line [7]. A retrospective study revealed that antibiotics improve the treatment efficacy of gemcitabine-based therapy in advanced cancer patients [8]. We hypothesized that improvement in the treatment efficacy of cytotoxic anticancer agents by adding antibiotics is independent of the tumor primary site but dependent on the type of anticancer agent. is retrospective study assessed whether antibiotics improve the treatment efficacy of oxaliplatin-based but not irinotecan-based therapy in advanced CRC patients.

Patients.
e medical records (2011-2018) of patients diagnosed with CRC histopathologically and administered oxaliplatin-or irinotecan-based therapy as first-or secondline treatment were retrospectively reviewed at the Department of Medical Oncology, Tohoku University Hospital, Japan. Inclusion criteria were as follows: (1) patients with histologically confirmed colorectal adenocarcinoma, (2) patients with least one measurable cancer lesion, (3) patients with unresectable or metastatic lesions, (4) patients who underwent at least one course of oxaliplatin-or irinotecanbased therapy, and (5) patients in whom the treatment efficacy of oxaliplatin-or irinotecan-based therapy had been assessed by computed tomography (CT) at least once. Patients who did not met these inclusion criteria were excluded from the study.
e study protocol was approved by the ethics committee of Tohoku University Hospital.

Treatment Methods.
e oxaliplatin-based therapy procedures in this study were as follows: (i) mFOLFOX6: 85 mg/m 2 of oxaliplatin and 200 mg/ m 2 of leucovorin given intravenously over 2 h, followed immediately by 400 mg/mg 2 of a fluorouracil (5-FU) intravenous bolus and then 2400 mg/ m 2 of 5-FU as a 46 h infusion (ii) SOX: 80 mg/m 2 of S-1 orally administered on days 1-14 and 130 mg/m 2 of oxaliplatin given intravenously on day 1 (iii) CapeOX: 1000 mg/ 2 of capecitabine orally administered twice a day on days 1-14 and 130 mg/m 2 of oxaliplatin given intravenously on day 1 e irinotecan-based therapy procedures were as follows: (i) mFOLFIRI: 150 mg/m 2 of irinotecan and 200 mg/ m 2 of leucovorin given intravenously over 2 h, followed immediately by 400 mg/mg 2 of a 5-FU intravenous bolus and then 2400 mg/m 2 of 5-FU given as a 46 h infusion (ii) S-1 plus irinotecan: 80 mg/m 2 of S-1 orally administered on days 1-14 and 125 mg/m 2 of irinotecan given intravenously on day 1 (iii) Irinotecan alone: 125 mg/m 2 of irinotecan given twice a week intravenously Both oxaliplatin group 1 and irinotecan group 1 were administered antibiotics from 2 weeks before the start of oxaliplatin-or irinotecan-based therapy, respectively, to the first imaging evaluation of the treatment efficacy of oxaliplatin-or irinotecan-based therapy, respectively, using CT.

Evaluation.
Responses were assessed using the Response Evaluation Criteria in Solid Tumors version 1.0 [9]. e complete response (CR; all signs of cancer disappeared after oxaliplatin-or irinotecan-based therapy) and partial response (PR; ≥30% decrease in the diameter of measurable lesions on CT) rates were combined and defined as the response rate (RR). e CR, PR, and stable disease (SD; <30% decrease and <20% increase in the diameter of measurable lesions on CT) rates were combined and defined as the disease control rates (DCR). e relative dose intensity of oxaliplatin-or irinotecan was defined as the ratio of the total actual dose to the planned dose. Hematological toxicity was reviewed from medical records and evaluated according to the Common Terminology Criteria for Adverse Events version 4.0 [10].

Statistical Analysis.
e median progression-free survival (PFS) and the median overall survival (OS) were calculated using the Kaplan-Meier method. P values of the RR and DCR between groups 1 and 2 were based on Fisher's exact test. Univariate and multivariate analyses were performed for the relationship between the response to oxaliplatin-or irinotecan-based therapy and the patients' background and severe neutropenia. Statistical analyses, including univariate analysis, multivariate analysis, Pearson's chi-square test, and Fisher's exact test, were performed using JMP ® 11 (SAS Institute Inc., Cary, NC, USA). P < 0.05 was considered statistically significant.

Patient Characteristics.
Patient characteristics are presented in Table 1. e number of patients who underwent oxaliplatin-based therapy as first-or second-line treatment was comparable between groups 1 and 2. Similarly, the number of patients who underwent irinotecan-based therapy as first-or second-line treatment was comparable between groups 1 and 2. e number of patients who underwent surgery for their primary lesion was significantly high in oxaliplatin group 2 compared to group 1 (Table 1). However, univariate and multivariate analysis showed that the imbalance in the ratio of patients who underwent surgery between oxaliplatin groups 1 and 2 does not influence the correlation between antibiotic treatment and the treatment efficacy of oxaliplatin-based therapy.

Efficacy of Oxaliplatin-or Irinotecan-Based
erapy. e RR and DCR of both oxaliplatin and irinotecan groups 1 and 2 are given in Table 2. Both RR and DCR were significantly high in oxaliplatin group 1 compared to oxaliplatin group 2. e RR of oxaliplatin groups 1 and 2 was 58.2% and 30.2%, respectively, while the DCR was 92.5% and 64.2%, respectively. In contrast, there was no significant difference in the RR and DCR between irinotecan groups 1 and 2. e RR of irinotecan groups 1 and 2 was 17.8% and 20.0%, respectively, while the DCR was 75.6% and 69.1%, respectively.
We divided oxaliplatin groups 1 and 2 into two groups, respectively. In each group, patients who were treated with fluoropyrimidine intravenously were assigned to oxaliplatin-1-mFOLFOX6 group and oxaliplatin-2-mFOLFOX6 group. In each group, patients who were treated with fluoropyrimidine orally were assigned to oxaliplatin-1-SOX/CapeOX group and oxaliplatin-2-SOX/CapeOX group. We compared    the response rate, median PFS and the median OS between oxaliplatin-1-mFOLFOX6 group and oxaliplatin-1-SOX/ CapeOX group or between oxaliplatin-2-mFOLFOX6 group oxaliplatin-2-SOX/CapeOX group, respectively. As shown in Supplemental Figure 1 and Supplemental Figure 2, there was no significant difference in median PFS or median OS between oxaliplatin-1-mFOLFOX6 and oxaliplatin-1-SOX/ CapeOX or between oxaliplatin-2-mFOLFOX6 group and oxaliplatin-2-SOX/CapeOX group, respectively.

Hematological Toxicity.
Hematological toxicity values of both oxaliplatin and irinotecan groups 1 and 2 are given in Table 3. e number of patients with severe leukopenia and neutropenia in oxaliplatin group 1 was significantly high compared to oxaliplatin group 2. e anemia and thrombocytopenia incidence rates and the increase in bilirubin, liver transaminase, and creatinine were similar in oxaliplatin groups 1 and 2. Similarly, the number of patients with severe leukopenia and neutropenia in irinotecan group 1 was significantly high compared to irinotecan group 2. e leukopenia, anemia, and thrombocytopenia incidence rates and the increase in bilirubin, transaminase, and creatinine were similar in irinotecan groups 1 and 2.

Univariate and Multivariate Analyses.
Results of univariate and multivariate analyses are shown in Table 4. We found a statistically significant relationship between the response to oxaliplatin-based therapy and antibiotic treatment (univariate analysis: P � 0.0159; multivariate analysis: P � 0.0114). e other seven factors were not significantly correlated with the response to oxaliplatin-based therapy. In addition, all eight factors were not significantly correlated with the response to irinotecan-based therapy.

Discussion
A previous study [7] revealed that a decrease in intratumor bacteria by antibiotic treatment augments the antitumor efficacy of gemcitabine in tumor-bearing mice. On the basis of that report [7], we retrospectively demonstrated that antibiotic treatment augments the treatment efficacy of gemcitabine-based therapy in advanced cancer patients [8]. In addition, a decrease in bacteria by adding antibiotics also augments the cytotoxicity of oxaliplatin but not of irinotecan in the CRC cell line in vitro [7], which is consistent with our results in this study. Antibiotic treatment is a factor that is significantly correlated with the efficacy of oxaliplatin-based therapy in advanced CRC patients.
Patients with leukopenia or neutropenia are generally administered antibiotics for prophylaxis [11]. erefore, it is inevitable that a high number of advanced CRC patients administered antibiotics get leukopenia or neutropenia compared to advanced CRC patients not administered antibiotics. is seems to indicate that adding antibiotics to oxaliplatin-or irinotecan-based therapy does not increase cytotoxicity by oxaliplatin-or irinotecan-based therapy in advanced CRC patients. In addition, there seems to be no correlation between an increase in the incidence rate of leukopenia and improvement in the treatment efficacy of anticancer agents. However, it has been reported that 5-FU and oxaliplatin combination therapy for patients with advanced colorectal cancer has stronger myelotoxicity than SOX therapy or CapeOX therapy for patients with advanced colorectal cancer [12,13]. ere was a higher rate of patients who were treated with mFOLFOX6 regimen in oxaliplatin group 1 when compared to those in oxaliplatin group 2. is might be one explanation to the reason why there were a higher rate of leukopenia and neutropenia in oxaliplatin group 1 than in oxaliplatin group 2.
It has been reported that the primary resection of colorectal cancer worsens the prognosis of patients with advanced colorectal cancer [14].
ere was a significantly higher resection rate of the primary site in oxaliplatin group 2 when compared to those in oxaliplatin group 1. e shorter overall survival time of oxaliplatin group 2 when compared to that of oxaliplatin group 1 might be partly attributable to the higher rate of the resection of the primary site in oxaliplatin group 2.
A previous study reported improvement in the treatment efficacy of anticancer agents by adding antibiotics in pancreatic cancer patients [8], indicating that this improvement is independent of the tumor primary site. In contrast, improvement in the treatment efficacy of anticancer agents by adding antibiotics seems to depend on the type of anticancer agent.
is study had a few limitations. First, the study had a retrospective design. Second, the number of patients included was relatively small. ird, cancer type studies were limited to CRC. Both gastric cancer and pancreatic cancer patients undergo oxaliplatin-based therapy in clinical practice [15,16]. In addition, lung cancer, gastric cancer, and ovarian cancer patients undergo irinotecan-based therapy [2,17,18]. However, we did not assess the treatment efficacy and safety of adding antibiotics to oxaliplatin-or irinotecanbased therapy in patients with these types of cancer. Fourth, we could not obtain data on the incidence rate of nonhematological toxicities such as diarrhea. ere are trillions of bacteria in the intestinal mucosa [19]. Antibiotic treatment decreases the number of bacteria in the intestinal mucosa and should augment the cytotoxicity of oxaliplatin in the intestinal mucosa, thereby increasing diarrhea. Fifth, we did not assess whether bacteria exist in tumor tissue pathologically in our patients. Fusobacterium nucleatum, which is part of the gut microbiome, is strongly associated with the tumorigenesis of CRC [20] and infiltrates cancer tissue in advanced CRC patients [21,22]. It is possible that in a large proportion of our patients, bacteria infiltrated CRC tissue. erefore, improvement in the treatment efficacy of oxaliplatin-based therapy might be attributable to a decrease in bacteria in tumor tissue by adding antibiotics. Further prospective or retrospective studies are required in order to overcome these limitations.
Journal of Oncology 5

Conclusion
Adding antibiotics is a potential therapeutic option to improve the treatment efficacy of oxaliplatin-based but not irinotecan-based therapy in advanced CRC patients. Prospective or retrospective studies to assess the treatment efficacy and safety of adding antibiotics to oxaliplatin-or irinotecan-based therapy are warranted.   e detailed retrospective observational data used to support the findings of this study are available from the first author (Hiroo Imai, e-mail: hiroo.imai.d8@tohoku.ac.jp) upon reasonable request. All data in the current study had no personal identifiers and were kept confidential.

Ethical Approval
is study protocol was approved by the ethics committee of Tohoku University Hospital.

Disclosure
All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflicts of Interest
Chikashi Ishioka, the corresponding author, received research funding from the Tokyo Cooperative Oncology Group, received contributions from Chugai Pharmaceutical, Ono Pharmaceutical, MSD, Pfizer, AstraZeneca, Bristol-Myers Squibb, Janssen Pharmaceutical, Taiho Pharmaceutical, Daiichi Sankyo Company, Limited, and Takeda Pharmaceutical, and is a representative of Tohoku Clinical Oncology Research and Education Society, a specified nonprofit corporation. Masanobu Takahashi received research funding from Ono Pharmaceutical.

Authors' Contributions
Hiroo Imai designed the study and wrote the initial draft of the manuscript. Chikashi Ishioka contributed to the analysis and interpretation of data and assisted in the preparation of the manuscript. All authors have contributed to data collection and interpretation and critically reviewed the manuscript. All authors approved the final version of the manuscript.

Supplementary Materials
Supplemental Table 1