Monitoring ctDNA RAS Mutational Status in Metastatic Colorectal Cancer: A Trial Protocol of RAS-trace and RAS-trace-2 Studies

Abstract

Background:

Spatial and temporal heterogeneities of RAS and other molecular genes should be considered in the treatment of metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs); acquired RAS mutation is sometimes observed at disease progression of treatment with the anti-EGFR mAb. At the same time, discrepancy of RAS status from tissues and circulating tumor DNA (ctDNA) in the same patient is sometimes observed. Based on this, we commenced two observational studies to clarify these heterogeneities of RAS and BRAF in mCRC, using next generation sequencing from liquid biopsy.

Methods/Design:

RAS-trace study is an observational study to monitor ctDNA RAS/BRAF/PIK3CA status every 4-12 weeks using the Plasma-SeqSensei™ CRC RUO Kit (Sysmex Inostics GmbH) in mCRC with RAS/BRAF wild-type (wt) on tumor tissue. The primary endpoint was the time to the acquired RAS mutations. A total of 42 patients has been accrued. RAS-trace-2 study is also an observational study aimed at comparing the efficacy of the anti-EGFR mAb in ctDNA RAS/BRAF wt with ctDNA RAS or BRAF mutant mCRC patients, whose RAS/BRAF are wt in tumor tissue. The primary endpoint was progression-free survival in patients with ctDNA RAS/BRAF wt and RAS or BRAF mutant. A total of 240 patients will be accrued over 2 years.

Discussion:

These trials will help us understanding the clinical significance of spatial and temporal heterogeneities of RAS, BRAF and other genes, while optimizing the anti-EGFR mAb treatment strategies in mCRC.

Background

Epidermal growth factor receptor (EGFR) is one of the key molecular targets in the treatment of metastatic colorectal cancer (mCRC). Mutation of RAS is a strong negative predictive factor of the anti-EGFR monoclonal antibody (mAb) efficacy in mCRC. PARADIGM study confirmed the superiority of panitumumab to bevacizumab combined with mFOLFOX6 in left-sided RAS wild-type (wt) mCRC[1]. Based on this, some guidelines have mentioned that standard first-line treatment of left-sided RAS/BRAF wt mCRC is an anti-EGFR mAb combined with mFOLFOX6/FOLFIRI[2,3].

Recently, growing evidence suggests that two heterogenetic aspects, temporal and spatial heterogeneities, should be considered in the treatment of mCRC[4]. Temporal heterogeneity implies that the status of RAS genes changes over time. It appears during treatment and may contribute to treatment resistance. Several trials reported that RAS mutation was detected in 30-40% of mCRC patients in a refractory setting, among those who had been previously treated with the anti-EGFR mAb[5,6]. This indicated that the emergence of RAS mutation occurred during the initial anti-EGFR mAb-based treatment in mCRC. However, limited data was available regarding the timing when the emergence of RAS mutation started and the frequency of acquired RAS mutation during first-line treatment using the anti-EGFR mAb. Spatial heterogeneity implies that the status of RAS genes varies from place to place. It can be seen within or between tumors. Several studies reported the discrepancy of RAS status in tumor tissues and liquid biopsy, and its clinical impact on mCRC patients treated with anti-EGFR mAb[6-8]. It was shown that there are dis-concordant rates of approximately 10% in the RAS mutations between primary tumor tissues and metastatic liver tissues[9]. ctDNA may be a good modality to overcome such spatial heterogeneity because it can depict the comprehensive genetic mutations in metastatic cancer patients with multiple metastatic sites.

To reveal the impact of temporal and spatial heterogeneity of RAS, and BRAF and other molecular genes on the efficacy of anti-EGFR mAbs in mCRC, we launched two trials (RAS-trace and RAS-trace-2) since 2022. Here, an overview of two trials and future treatment strategies using liquid biopsy are presented.

Methods/Design

Ras-trace

RAS-trace is a pilot study to monitor the ctDNA status of RAS, BRAF, and PIK3CA, using the Plasma-SeqSensei™ CRC RUO Kit (Sysmex Inostics GmbH) for RAS/BRAF wt (diagnosed from tissue samples) and mCRC. Plasma-SeqSensei™ CRC RUO Kit is an NGS-based method using Safe-SeqS technology[10] that enables highly sensitive detection of ctDNA (Figure 1). The key eligibility criteria are as follows: histologically-proven RAS/BRAF wild mCRC, cStage IV or recurrent CRC, more than 12 cycles of chemotherapy (approximately 24 weeks or more) including anti-EGFR mAb is planned, measurable lesions, with no previous history of chemotherapy and radiotherapy, aged 20 years old and over, and ECOG PS0-1. Active multiple and double cancers were excluded. ctDNA RAS/BRAF/PIK3CA were evaluated at baselines (before chemotherapy), 8, 12, 16, 20, 24, 36, 48 weeks and disease progression, with next generation sequencing. Further ctDNA examinations are not conducted once patients experience disease progression. The primary endpoint is the time to the acquired RAS mutations. The second endpoints consisted of correlation between the time to RAS mutations acquired, frequencies of BRAF/PIK3CA mutations in ctDNA, progression-free survival (PFS), and overall survival (OS). The total sample size was 40. This study commenced in Jan 2022, involving seven Japanese institutions, and completed accrual in May 2023. Primary analysis is planned for 2024.

Figure 1.

Trial Schema of RAS-trace.

RAS-trace-2

RAS-trace-2 is an observational study to compare the efficacy of the anti-EGFR mAbs in ctDNA RAS/BRAF wt with RAS or BRAF mutant (mt) mCRC patients whose tissue-derived RAS/BRAF are wt (Figure 2). The Plasma-SeqSensei™ Solid Cancer IVD Kit (Sysmex Inostics GmbH) was also used for evaluation of ctDNA RAS/BRAF. The key eligibility criteria are similar to RAS-trace. Deficient mismatch repair (dMMR) was excluded. RAS/BRAF/PIK3CA was evaluated at baseline, and disease progression. The primary endpoint was PFS in patients with ctDNA RAS/BRAF wt and RAS or BRAF mt. Secondary endpoints included the response rate (based on RECIST ver1.1) in ctDNA RAS/BRAF wt and RAS or BRAF mt, OS, adverse events, frequency of ctDNA mutation, and exploratory analysis of resistant genes for anti-EGFR mAb. The sample size was calculated as 240 patients in total, assuming 13.7 months of PFS in ctDNA RAS/BRAF wt and 9.0 months in ctDNA RAS or BRAF mt with a one-sided α level of 5%, power of 70%, an accrual period of 2 years, and a follow-up period of 3 years. PFS of 13.7 months was set according to that in RAS wt mCRC patients treated with mFOLFOX6+Panitumumab in the PARADIGM study[1], while PFS of 9.0 months was set according to that in RAS wt (tissue confirmed) mCRC patients with ctDNA RAS mt in the FIRE-4 study[7]. This sample size was estimated assuming that approximately one-sixth patients are ctDNA RAS or BRAF mutant. This proportion was monitored and modification of sample size is planned once the observed proportion is far from one-sixth. It was recommended that CT scan and tumor marker test (CEA and CA19-9) were conducted every 8-12 weeks and 4 weeks, respectively. This study commenced in June 2023 and involved 17 leading Japanese institutions.

Figure 2.

Trial Schema of RAS-trace-2.

Discussion

In current practice, anti-EGFR mAbs are administered based on the RAS mutational status of tumor tissues, mostly primary tumors in CRC. Therefore, in some cases, anti-EGFR mAbs are given to CRC patients with multiple liver metastases, whose RAS status is mutant. Moreover, the anti-EGFR mAb combined with mFOLFOX6/FOLFIRI as a first line treatment for mCRC are administered until disease progression, however the resistance to the anti-EGFR mAb may be acquired before the patients become refractory to the mFOLFOX6/FOLFIRI in some mCRC patients. Our platform will provide some answers to spatial and temporal heterogenetic aspects of mCRC.

Currently, the OncoBEAM™RAS CRC kit (Sysmex Corporation) is commercially available to monitor the RAS status in mCRC[11]. This is used when an anti-EGFR mAbs rechallenge is planned. The REMMARY/PURSUIT trials, aiming for the efficacy of rechallenge with the anti-EGFR mAb, showed that no response of anti-EGFR mAbs was observed among 7 patients with ctDNA RAS mutation at progression on prior anti-EGFR mAb[12]. This indicated that ctDNA RAS testing at progression on prior use of the anti-EGFR mAb may predict the efficacy of rechallenge with the anti-EGFR mAb. Furthermore, as mentioned previously, molecular alternation of RAS is ongoing during first-line treatment with anti-EGFR mAbs. Although there are several genomic alterations which may link to anti-EGFR therapy inhibition aside from RAS, it can be reasonable to switch from anti-EGFR therapy to anti-VEGF therapy when the emergence of mutations in RAS is observed during first-line treatment in RAS wt mCRC. RAS-trace may provide new evidence regarding the timing when acquired RAS mutations were observed with their clinical impact on the efficacy of anti-EGFR mAbs in mCRC.

The aspect from spatial heterogeneity should also be considered in treatment of mCRC. In the PARADIGM trial, among histologically confirmed RAS wt mCRC patients, 7.4% exhibited ctDNA RAS mutation at baseline liquid biopsy[8]. Baseline liquid biopsy data of the FIRE-4 trial, which aimed to confirm the efficacy of early switch maintenance from cetuximab to bevacizumab during first-line therapy in RAS wt CRC, was recently presented[7]. Among all the registered patients who with confirmed RAS wt tumor tissues, the frequency of ctDNA RAS mutation was 13%. Surprisingly, registered mCRC patients with ctDNA RAS mt had a shorter PFS and OS than those with ctDNA RAS wt. Our RAS-trace-2 clarifies the role of liquid biopsy at baseline in the treatment of mCRC patients who are diagnosed as RAS wt from resected tumor tissue samples.

Involvement of other molecular biomarker resistance to anti-EGFR mAbs should be considered. Aside from RAS, BRAF V600E, several biomarkers have already been known as negative predictive biomarkers of anti-EGFR mAbs[13]. The prognostic impact of emergence of these biomarkers, and alternation of these biomarkers at disease progression, were not fully evaluated. These analyses are planned in our exploratory trial platform.

In this article, an overview of our challenges was presented. Our studies will reveal the clinical relevance of liquid biopsy in the verification of RAS/BRAF mutational status, and provide an optimal treatment strategy which can maximize the effect of anti-EGFR mAbs in mCRC.

Conflicts of Interest

KK received lecture fees from Merck, Takeda, and Eli Lilly, and research funds from Sysmex Corporation. JF is an employee of the Sysmex Inostics GmbH. MS received lecture fees from Merck, Takeda, Yakult, Ono, and Eli Lilly. JW received lecture fees from Eli Lilly and Takeda. TK received lecture fees from Daiichi Sankyo, Taiho, Bristol-Myers Squibb, Ono, and Eli Lilly. NM received lecture fees from Takeda and scholarship endowments from Yakult. KY received lecture fees from Merck, Takeda, Chugai, Taiho, Yakult, Ono, Eli Lily, MSD and Bristol. KM received lecture fees from Chugai, Ono, Daiichi Sankyo and Eli Lilly. The rest of the authors do not have any conflicts of interest.

Source of Funding

RAS-trace and RAS-trace-2 received financial support from Sysmex Corporation and Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering.

Author Contributions

We have twenty-two authors but all the authors have reviewed the manuscript critically, have contributed significantly, and are in agreement with the content of the manuscript. Especially, Kozo Kataoka, Takeshi Yamada, Kentaro Yamazaki, Johannes Fredebohm, Manabu Shiozawa and Masataka Ikeda contributed to the conception and design of this study. Keita Mori mainly contributed to the statistical analysis. Masataka Ikeda, The rest of authors (Naoto Takase, Kazuma Ito, Jun Watanabe, Toshihiro Kudo, Takeshi Suto, Toshihiko Matsumoto, Kohei Murata, Yusuke Suwa, Shogen Boku, Hisateru Yasui, Nobuhisa Matsuhashi, Atsuyuki Maeda, Kiichi Sugimoto, Yusuke Matsumoto, Mitsuru Yokota) mainly contributed to the acquisition of the data.

Approval by Institutional Review Board (IRB)

RAS-trace and RAS-trace-2 were approved by IRB in Hyogo Medical University (No. 3951 and 4409).

Disclaimer

Takeshi Yamada is one of the Associate Editors of Journal of the Anus, Rectum and Colon and on the journal's Editorial Board. He was not involved in the editorial evaluation or decision to accept this article for publication at all.

Trial registration information

RAS trace: jRCT1050210160, RAS-trace-2: jRCT1050230049.