The development of combination therapy has prolonged the median survival length of patients with advanced colorectal cancer (CRC) to more than 25 months (1). A common treatment strategy consists of combination therapy with a fluoropyrimidine plus oxaliplatin or irinotecan (FOLFOX or FOLFIRI) as first-line treatment, followed by a second-line treatment using the regimen not selected in the first-line treatment. Evidently, ‘the use of all three active drugs in advanced CRC produces the longest overall survival (OS)’ (2). In addition, the introduction of molecular-targeted drugs, such as anti-vascular endothelial growth factor and anti-epidermal growth factor receptor (EGFR) antibodies, has further improved the prognosis of patients with metastatic CRC (1).

Continuation of bevacizumab, an anti-vascular endothelial growth factor antibody, in the second-line treatment after first-line combination therapy with a conventional cytotoxic doublet and bevacizumab is a widely accepted clinical practice (3). However, the efficacy and safety of the uninterrupted use of an anti-EGFR antibody, such as panitumumab, in the second-line treatment after disease progression during first-line treatment with anti-EGFR antibody remains to be determined. In contrast to bevacizumab, which elicits its anti-tumor effect only in combination with cytotoxic agents, anti-EGFR antibody alone has clinical benefits in tumor response, OS, and progression-free survival (PFS) (4). Thus, the progression of disease during first-line treatment with cytotoxic doublets and anti-EGFR antibody suggests the development of resistance against both agents to various degrees.

Nevertheless, the potential benefit of uninterrupted (continuous) use of anti-EGFR antibody in second-line therapy cannot be denied, especially when irinotecan-based regimens are saved for second-line therapy. A randomized controlled trial was conducted to compare anti-EGFR monotherapy (cetuximab) and irinotecan plus anti-EGFR combination therapy in 329 patients whose disease had progressed during or within 3 months after irinotecan-based treatment (5). The authors reported that the response rate in patients with combination therapy was significantly higher than that observed during irinotecan monotherapy (22.9 vs. 10.8%, P=0.007). The median time to progression was also significantly longer when combination therapy was administered (4.1 vs. 1.5 months, P<0.001). The result suggests that cetuximab influences the mechanism enhancing the effect of irinotecan (5). Specifically, the authors speculated from preclinical laboratory studies that the inhibition of EGFR by cetuximab may have disabled drug efflux (6), augmented apoptosis induced by irinotecan (7), or weakened DNA-repair activity (8), resulting in the enhancement of the effect derived from irinotecan.

However, the efficacy and safety of the continuous use of panitumumab in first- and second-line treatments for CRC have yet to be determined. We consider that the present study, which addresses this point, will contribute to the further understanding of tumor biology and the improvement of second-line treatment strategies.

Even in patients with RAS wild-type CRC, the clinical efficacy of anti-EGFR antibodies is limited by the development of acquired (secondary) resistance (9). Preclinical models have shown that some alterations in genetic status allow selective tumor cells to grow under anti-EGFR therapy, which raises interest about whether or not these mechanisms are also active in clinical samples. However, analyzing pre- and post-treatment tissue to clarify this question has been hampered due to the invasive nature of the biopsy procedure, which is often unnecessary for individual patients. In addition, identifying the mechanisms of resistance would be quite difficult in view of the intra-tumor heterogeneity, because multiple biopsies within the tumor of interest would be required.

As a breakthrough for this problem, the measurement of circulating tumor DNA (ctDNA), termed ‘liquid biopsy’, is gaining attention. First, a study revealed that tumor-related ctDNA was found in the majority of patients with metastatic CRC. In addition, the analysis of tumor-related ctDNA can identify a specific mutant genotype, and provide an estimation of total tumor burden (10). Furthermore, Diaz et al found that one third of the patients developed multiple different KRAS mutations in their sera between 5 and 6 months following treatment, even though their tumors were initially KRAS wild-type. This result suggests that mutant KRAS genes are released into the circulation with the emergence of resistance to anti-EGFR antibodies (11).

The measurement of ctDNA enables us to continuously monitor gene mutation status, and may predict the clinical benefit of ongoing anti-EGFR antibody therapy. However, KRAS mutations in ctDNA were only identified in a small subset of patients, leaving the mechanism of acquired resistance unexplained. Thus, the present study will investigate the mutation status of other tumor-related genes, including extended RAS genes. The results should provide additional information regarding the mechanism of acquired resistance of CRC against anti-EGFR antibodies, and ultimately lead to the establishment of personalized cancer treatment.

This study is a multicenter single-arm Phase II clinical trial. The objective is to evaluate the effect of FOLFIRI with panitumumab as the second-line chemotherapy in patients with advanced and/or recurrent CRC, after failure of the first-line treatment with FOLFOX with panitumumab, using the 6-month PFS rate as the primary endpoint. OS, PFS, time to treatment failure, overall response rate, disease control rate, safety, and gene mutation status will be evaluated as secondary endpoints (Table I).

Patients who meet all of the inclusion criteria and none of the exclusion criteria listed below will be eligible for enrollment in this study.

Inclusion criteria are: a) Histologically-confirmed inoperable colorectal adenocarcinoma excluding vermiform appendix cancer and anal canal cancer; b) Refractory tumor with measurable disease according to the Response Criteria in Solid Tumors (version 1.1) (12) that has progressed, or the appearance of an evaluable new lesion after first-line therapy with FOLFOX plus panitumumab; c) age ≥20 years at the time of informed consent; d) Eastern Cooperative Oncology Group performance status of 0–2; e) written informed consent prior to study-specific screening procedure; f) life expectancy of at least 90 days; g) withdrawal from first-line chemotherapy using FOLFOX plus panitumumab for RAS wild-type advanced or metastatic CRC due to intolerable toxicity (but not to panitumumab alone) or progressive disease; h) adequate organ function according to the following laboratory values obtained within 14 days before enrollment (excluding patients who received blood transfusions or hematopoietic growth factors within 14 days before the laboratory test): i) Neutrophil count: ≥1,500/mm³, ii) platelet count: ≥10.0×10⁴/mm³, iii) hemoglobin: ≥8.0 g/dl, iv) total bilirubin: ≤1.5 mg/dl, v) aspartate aminotransferase, alanine aminotransferase: ≤100 IU/l (≤200 IU/l if liver metastases present), and vi) serum creatinine: ≤1.5 mg/dl; and h) confirmed RAS wild-type tumor at the time of first-line therapy

Exclusion criteria are: a) History of other malignancy with a disease-free interval of <5 years (other than curatively-treated cutaneous basal cell carcinoma, curatively-treated carcinoma in situ of the cervix, and gastroenterological cancer confirmed to be cured by endoscopic mucosal resection); b) massive pleural effusion or ascites requiring intervention; c) radiological evidence of brain tumor or brain metastases; d) active infection including hepatitis; e) any of the following concurrent diseases: i) gastrointestinal bleeding or obstruction (including paralytic ileus), ii) symptomatic heart disease (including unstable angina, myocardial infarction, or heart failure), iii) interstitial pneumonia or pulmonary fibrosis, iv) uncontrolled diabetes mellitus, and v) uncontrolled diarrhea (that interferes with daily activities despite adequate therapy); and f) any of the following medical history: i) myocardial infarction: History of one episode within one year before enrollment or two or more lifetime episodes, ii) serious hypersensitivity to any of the study drugs, iii) history of adverse reaction to fluoropyrimidines suggesting dihydropyrimidine dehydrogenase deficiency, iv) previous treatment with irinotecan hydrochloride, v) current treatment with atazanavir sulfate; vi) previous treatment with tegafur, gimeracil, and oteracil potassium within seven days before enrollment, vii) pregnant and lactating females, and males and females unwilling to use contraception, viii) requires continuous treatment with systemic steroid, ix) psychiatric disability that would preclude study compliance, and x) Otherwise determined by the investigator to be unsuitable for participation in the study.

Treatment with the FOLFIRI and panitumumab regimen will be continued in 2-week cycles (Table II) until disease progression, unacceptable toxicity and/or patient refusal. Dose reduction will be carried out according to the protocol (Table III).

The study will be conducted according to the approved protocol and in compliance with the principles of the Declaration of Helsinki, International Conference of Harmonization-Good Clinical Practice, and the ‘Ethical Guidelines for Medical and Health Research Involving Human Subjects’, and comply with the regulations in Japan. The Institutional Review Board of the Epidemiological and Clinical Research Information Network (17-0601-1) approved the protocol. The protocol and patient information sheet used to conduct the study must be approved by the Institutional Review Board at each study site prior to the start of study participation. After the study has been explained with sufficient time for consideration, if the patient voluntarily agrees to participate in the study, written informed consent will be obtained.

The study is conducted with the Epidemiological and Clinical Research Information Network-related study group and financially supported by Takeda Pharmaceutical Co., Ltd. Although the study will be conducted with drugs manufactured and marketed by Takeda Pharmaceutical Co., Ltd., the company will not play any role or have any authority in the analysis of results, interpretation, report writing, or decisions for data disclosure.

FOLFIRI with panitumumab is recognized as one of the standard second-line therapies for advanced and/or recurrent CRC. Clarifying the benefit and safety of continuous administration of panitumumab with FOLFILI after tumor progression during first-line FOLFOX plus panitumumab treatment will further improve second-line treatment strategies. Evaluation of gene mutation status by liquid biopsy will provide distinctive evidence concerning treatment efficacy and unveil the mechanism underlying resistance development. Ultimately, the information gained from this study is expected to lead to the establishment of a biomarker for tumor response during second-line therapy with panitumumab. The results of the present study will be disseminated at related international congresses and in peer-reviewed journals.