Abstract
Background/Aim: Secondary mutation of mutated RAS, induced by chemotherapy, is thought to be rare. However, introduction of liquid biopsy (LB) has made it possible to monitor RAS status in patients’ plasma throughout the course of chemotherapy for metastatic colorectal cancer (mCRC), and disappearance of the RAS mutation (RAS-mt), i.e., the NeoRAS-wt phenomenon, has been reported and is receiving attention, especially with respect to treatment implications. Patients and Methods: A prospective study of 129 patients undergoing chemotherapy for mCRC (RAS-wt, n=65; RAS-mt, n=64) was carried out. Plasma RAS status was monitored in these patients by LB. Relations between secondary genetic change, chemotherapy, and 6-month disease outcomes were analyzed. The effect of anti-EGFR mAb therapy on NeoRAS-wt mCRC was also examined. Results: NeoRAS-wt was detected in 27 (43.5%) RAS-mt patients overall and in all patients with a G12S or Q61H mutation. First-line treatment was more effective among NeoRAS-wt patients than non-NeoRAS-wt patients (70.9% vs. 48.6% overall response rate, p=0.087), and the time from treatment to LB was shorter in this group. Six-month outcomes were significantly better in the NeoRAS-wt group (p<0.001), and conversion to NeoRAS-wt was found to be predictive of a good outcome (OR=7.886, 95% CI=2.458-25.30; p<0.001). Anti-EGFR mAb therapy was found to restrict disease progression in NeoRAS-wt patients. Conclusion: Conversion to NeoRAS-wt is relatively frequent, and it may predict good responses to treatment. Anti-EGFR mAb therapy was effective for our NeoRAS-wt patients. Detection of NeoRAS-wt by LB may significantly change the indication for anti-EGFR mAb therapy and the mCRC treatment strategy.
Metastatic colorectal cancer (mCRC) requires systemic chemotherapy. Such treatment has remained substantially unchanged over the past 10 years, generally involving administration of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) or anti-vascular endothelial growth factor (VEGF) mAb in addition to oxaliplatin, irinotecan, and 5-fluorouracil (1-3). Generally, before a decision is made to initiate anti-EGFR mAb therapy, RAS genotyping is performed. A RAS mutation (RAS-mt) is detected in approximately 50% of all mCRCs (4, 5), and current guidelines restrict administration of anti-EGFR mAb to patients without RAS-mt mCRC, i.e., to patients with wild-type RAS (RAS-wt) mCRC (1-3). Several studies have shown survival of patients with mCRC and RAS-mt detected in tissue (RAS-mt mCRC) to be shorter than that of patients with RAS-wt mCRC; use of anti-EGFR antibodies in combination with chemotherapy significantly increased efficacy of treatment in patients with RAS-wt mCRC but did not benefit patients with RAS-mt mCRC (6-8).
Tumor heterogeneity refers to the notion that a single tumor consists of numerous subclone cells, with the subclonal diversity being indicative of the mechanism by which cancer cells acquire individual resistance (9, 10). Selection of subclones during chemotherapy is thought to induce significant oncologic changes and variation in the response to treatment, but the mechanism for selection, including RAS clones in mCRC is not well understood (11, 12).
Genotyping of RAS mutational status in patients with mCRC has typically been tissue-based, but recently liquid biopsy (LB), performed with the OncoBEAM™ RAS CRC Kit (Sysmex Corporation), has made it possible to confirm RAS status in circulating tumor DNA isolated from plasma and to frequently monitor RAS status during treatment (13, 14). Plasma analysis brought to light conversion from RAS-wt mCRC to RAS-mt mCRC during chemotherapy together with anti-EGFR mAb therapy (15). Several investigators have indicated that emergent RAS mutations may drive clonal selection and thus underlie acquired resistance to anti-EGFR therapy and have shown further that such emergent mutations are predictive of a reduced benefit from such therapy and even of a poor prognosis (15-18). With the exception of conversion brought about by anti-EGFR antibody therapy for RAS-wt mCRC, secondary RAS mutations are thought to be rare (19, 20).
The possibility of chemotherapy-induced reversion of RAS-mt mCRC to RAS-wt (NeoRAS-wt) mCRC, the so-called NeoRAS-wt phenomenon, has come to be advocated. Henry et al. (21), at the 2020 Gastrointestinal Cancers Symposium, reported the phenomenon but described it as rare (occurring in 8% of cases). Moati et al. (22) also reported that the frequency of NeoRAS-wt was only about 5%, and Klein-Scory et al. (23) reported that RAS-mt disappeared rapidly during first-line treatment and converted to RAS-wt in more than 90% (10/11) of patients with tissue RAS-mt mCRC who responded to first-line treatment.
NeoRAS-wt is attracting attention because new treatments are needed to increase therapeutic options for patients with RAS-mt mCRC. Specifically, treatment-induced conversion from mutant RAS to wild-type RAS raises the question of whether indications for anti-EGFR mAb therapy change in patients with mCRC for which RAS mutation status changes during chemotherapy. Herein, the clinical question is as follows: Will administration of anti-EGFR mAb prove to be a useful tool for control of mCRC in patients in whom RAS-mt alleles are being replaced by NeoRAS-wt alleles? In this regard, we conducted a prospective observational study in which we examined the frequency of reversion to NeoRAS-wt, identified clinical characteristics associated with reversion to NeoRAS-wt, and investigated the usefulness of anti-EGFR mAb therapy for NeoRAS-wt mCRC.
Patients and Methods
Study participants. Included in this study were 129 patients who were receiving treatment for unresectable mCRC at Kanagawa Cancer Center between September 2020 and March 2021 and in whom RAS status had been determined in tumor tissue (tissue RAS). All had completed at least one chemotherapy regimen. A prospective database was set up to store patients’ clinical characteristics, tumor characteristics, responses to treatment and side effects. Treatments were administered until disease progression, occurrence of major toxicity, secondary surgery, or death. In principle, the choice of therapeutic regimen, including treatment after a patient’s disease was determined to be progressive, were discussed and decided on weekly conferences that involved a multidisciplinary team. Postponement of chemotherapy and/or dose reduction was left to the discretion of each patient’s physician. RAS mutational status in plasma (plasma RAS) was tested by LB at the time of routine pre-chemotherapy blood tests. LB was performed with the use of OncoBEAM, according to the manufacturer’s instructions.
Patients were enrolled in the study while under chemotherapy, having provided informed consent for their participation. The study was approved by the institutional review board of the Kanagawa Cancer Center (Approval number: 2020EKI-121).
Clinical assessments. The patients’ clinical characteristics (sex, age, body mass index), laboratory values, results of pathological examination, the type and course of chemotherapy, and the response to chemotherapy were obtained from our hospital’s electronic health records, and we examined their background. The response of each patient’s mCRC to treatment was assessed 6 months after LB by the physician in charge, radiologists, and data managers in accordance with RECIST guidelines (version 1.1) (24); it was classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) on the basis of computed tomography images.
A flow diagram of the study is shown in Figure 1. Patients were divided into 4 groups according to tissue RAS and plasma RAS status, and response to treatment 6 months after LB was evaluated on a per group basis.
Study flow diagram. One hundred twenty-nine patients with unresectable metastatic colorectal cancer (mCRC) were divided into 2 groups on the basis of tissue RAS status. They were then further divided into 4 groups on the basis of plasma RAS status determined by means of liquid biopsy (LB). Disease outcomes were assessed 6 months after LB. Pts: Patients.
Statistical analysis. Continuous variables are shown as median [interquartile range (IQR)] values, and categorical variables are shown as the number (percentage) of patients, unless otherwise indicated. Differences in continuous variables were analyzed by the Wilcoxon/Kruskal-Wallis test and differences in categorical variables by Fisher’s exact test. Haberman’s residual analysis (25), which allows for the significance of each criterion to be calculated independently, was used to determine significant differences in mutated allele variants in tissue RAS-mt patients. Adjusted standardized residuals (ASRs) were calculated, and an ASR >1.96 was taken to indicate a significant difference from expected values. Multiple regression modeling was used to identify factors that were independent predictors of disease progression. Factors entered into the model were chosen a priori from those reported in the literature (26-31). As the number of observations was small compared to the number of factors, linear regression was prone to overfitting. Least absolute shrinkage and selection operator (LASSO) regression was applied to prevent overfitting (32) and to select factors potentially predictive of disease progression. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. All statistical analyses were performed with JMP Pro 15 (SAS Institute Inc., Cary, NC, USA), and p<0.05 was considered significant for all.
Results
The total study group comprised 70 men and 59 women aged 68 years (28 to 88 years). Sixty-five patients had tissue RAS-wt mCRC and 64 had tissue RAS-mt mCRC. The primary lesion was resected in almost all patients. Because anti-EGFR mAb could not be used in the tissue RAS-mt patients, the tissue RAS-wt and RAS-mt groups differed in terms of the first-line treatment regimen, but other clinical variables did not differ significantly between these 2 groups (Table I).
Patient and tumor characteristics (N=129) and first-line treatment, per RAS mutational status in tissue.
Results of LB are shown per group in Table II. On LB, plasma RAS-mt was detected in 12 (18.5%) of the 65 tissue RAS-wt patients. All 12 of these patients had received anti-EGFR mAb therapy. Among the 64 tissue RAS-mt patients, plasma RAS-wt was found in 27 (42.2%). There was no statistical between-group difference in the number of chemotherapy lines or types of drugs received by patients before LB.
Results of liquid biopsy for plasma RAS status and clinical variables at the time of examination, per tissue RAS status.
We investigated characteristics of 62 of the 64 RAS-mt patients. Two patients for whom LB results were unclear were excluded from this investigation. We divided the tissue RAS-mt patients into 2 groups based on plasma RAS status determined by LB. Thus, as shown in Table III, the 2 groups comprised 27 NeoRAS-wt patients and 35 non-NeoRAS-wt patients. There was no statistical difference between these 2 groups in the location of the primary tumor, resection of the primary tumor, histologic type, frequency of synchronous metastases, or sites of metastasis. However, the primary tumor was resected in all patients (p=0.119) in the NeoRAS-wt group, and metastasis confined solely to the lung tended to be more prevalent in this group (p=0.012). Types of chemotherapy plus types of mAb therapy, used as first-line treatment, and related variables are shown for the NeoRAS-wt group and non-NeoRAS-wt group in Table IV. There was no between-group difference in the types of chemotherapy used for first-line treatment, but the response to first-line treatment was much better in the NeoRAS-wt group than in the non-NeoRAS-wt group, with the overall response rate being more than 70% (vs. 48.6% in the non-NeoRAS-wt group). Drugs administered prior to LB did not differ significantly between the 2 groups, and the number of lines of chemotherapy received by patients was similar between groups, but time from the start of first-line treatment to LB was significantly shorter in the NeoRAS-wt group than in the non-NeoRAS-wt group [342 (188-637) days vs. 629 (315-1,008) days, respectively; p=0.046] (Table IV).
Characteristics of tissue RAS-mt patients for whom liquid biopsy results were clear (N=62) and association with plasma RAS mutation status.
Chemotherapy and related variables, per NeoRAS-wt vs non-NeoRAS-wt.
To identify other factors associated with NeoRAS-wt, we investigated mutated allele variants in tissue RAS-mt patients (Table V) and found a change from RAS-mt to NeoRAS-wt in all patients with G12S and Q61H mutation. On the other hand, G13D tended to be less negative (30.0%). On assessment of ASRs, we found that the ratio of G12S and Q61H was significantly different from that of other variants (p=0.048 and 0.023, respectively).
Mutated allele variants in tissue RAS-mt patients (N=62) in relation to plasma RAS mutation status.
Disease outcomes 6 months after LB are shown in Figure 2. In the non-NeoRAS-mt group, 24 patients (68.6%) including the 12 (34.3%) who died, were shown to have PD, whereas, in the NeoRAS-wt group, 5 patients (18.5%), including the 1 (3.7%) who died, were shown to have PD. The relatively good outcomes in the NeoRAS-wt group were statistically significant (p<0.001). Outcomes were also significantly better in the tissue RAS-wt with plasma RAS-wt group than in the tissue RAS-wt with plasma RAS-mt group (p=0.129).
Bar graph showing disease outcomes 6 months after liquid biopsy (LB), per study group. PR: Partial response; SD: suitable disease; PD: progressive disease; BSC: best supportive care. Number (percentage) of patients is shown.
A poor response to first-line chemotherapy (26), tumor containing poorly or undifferentiated (vs. well or moderately differentiated) tissue components (27, 28), synchronous (vs. metachronous) metastasis (29), and a right-sided (vs. left-sided) primary tumor (30, 31) have been identified previously as predictive of a poor outcome in cases of mCRC. Thus, in addition to NeoRAS-wt vs. non-NeoRAS-wt, we entered these variables into a LASSO regression model. Non-NeoRAS-wt was the only factor found to be an independent predictor of disease progression 6 months after LB in the tissue RAS-mt patients (OR=7.886, 95% CI=2.458-25.30; p≤0.001) (Table VI).
Results of LASSO regression analysis performed to identify risk factors for disease progression in tissue RAS-mt mCRC patients.
Four of our NeoRAS-wt cases were treated with cetuximab and panitumumab as anti-EGFR mAb therapy (Table VII). Outcomes in these cases were PR (N=1), SD (N=2), and PD (N=1). The responses to treatment including anti-EGFR mAb therapy were good in 3 of the 4 cases, and the anti-EGFR mAb therapy was continued in the respective patients for over 6 months without any signs of severe toxicity.
Cases of conversion to NeoRAS-wt for which anti-EGFR mAb therapy was applied.
Discussion
With LB assays having become commercially available, use of anti-EGFR antibody in the treatment of mCRC has changed greatly. It can be said that LB has greatly influenced indications for anti-EGFR antibody therapy. RAS gene mutations have been shown to be equally prevalent in both primary and metastatic lesions (19), with a meta-analysis yielding a matching rate of 93% (33). Secondary RAS gene mutations are rare in patients having undergone chemotherapy without the addition of anti-EGFR antibody therapy (20). However, it has since been reported that RAS mutant clones develop after treatment of RAS-wt mCRC with anti-EGFR mAbs (34) and that re-confirmation of RAS-wt by LB after development of RAS-mt is useful prior to reuse of anti-EGFR antibodies for patients with plasma RAS-wt mCRC (34, 35). The Neo-RAS wt phenomenon, i.e., change from RAS-mt to RAS-wt during chemotherapy, is an established phenomenon, but the characteristics and incidence of NeoRAS-wt as well as effects of anti-EGFR mAb for NeoRAS-wt patients have been unclear.
The study reported herein overturned the conventional wisdom regarding RAS status, i.e., the notion that secondary RAS mutations are rare except in patients who have received anti-EGFR antibody therapy for RAS-wt mCRC. The NeoRAS-wt phenomenon is seen in clinical practice. In fact, 42% of patients in our tissue RAS-mt group who did not receive anti-EGFR mAb therapy showed a change to RAS-wt, that is, NeoRAS-wt.
As noted above, change to NeoRAS-wt was found not to be directly related to the location of the primary tumor, its histological type, drugs used before LB, and the number of chemotherapy regimens received. Rather, change to NeoRAS-wt was significantly associated with a good response to first-line chemotherapy. We speculate that the positive response to chemotherapy was an important and perhaps critical factor responsible for the change to NeoRAS-wt. G12S and Q61H mutations may be linked to the change to NeoRAS-wt, but an accumulation of cases is needed before we can explore this possibility in depth.
In the study described herein, we examined outcomes after LB. NeoRAS-wt tended to be detected sooner (after the start of treatment) than non-NeoRAS-wt, and at 6 months after detection, the mCRC was found to be better controlled in 81.5% of our NeoRAS-wt patients compared to non-NeoRAS-wt; 31.4%. Interestingly, it was confirmed that the RAS status had changed a second time, this time from NeoRAS-wt to RAS-mt at the time PD was documented in the 4 patients in the NeoRAS-wt group whose disease worsened. Furthermore, considering the fact that PD was documented 6 months after LB in about 70% of patients in the non-NeoRAS-wt group, including 34% of those who died, it appears that it may be possible to predict a good outcome on the basis of NeoRAS-wt and that plasma RAS-mt may be useful as a predictor of disease exacerbation.
Subsequent treatment for NeoRAS-wt patients with anti-EGFR mAb is increasingly being discussed. To investigate its effect, we used anti-EGFR mAb in NeoRAS-wt cases, namely, in patients in whom RAS-mt was originally detected and then RAS-wt was detected after chemotherapy. The responses to the anti-EGFR mAb treatment were good, even after previous treatment was followed by PD, and these patients were able to remain on anti-EGFR mAb therapy for more than 6 months without development of severe toxicity. The indication for anti-EGFR mAb treatment remains controversial, but outcomes in 3 recent cases reported by Osumi et al. (36), 7 recent cases reported by Bouchahda et al. (37), and 4 recent cases reported by Raimondi et al. (38) showed that some patients benefit from anti-EGFR mAb therapy. Results in these cases and in ours imply that anti-EGFR mAb therapy is effective for NeoRAS-wt mCRC, prolonging overall survival of patients with tissue RAS-mt mCRC.
Investigation into NeoRAS-wt is in its early stage. Gazzaniga et al. (39) reported in 2018 a case in which anti-EGFR mAb was useful. In 2019, Chibaudel et al. (40) reported that tissue RAS-mt could be converted to RAS-wt in 50% of patients by treatment with anti-VEGF mAb, and a prospective multicenter single-arm open-label phase II study (CETIDYL study) aimed at determining the effect of anti-EGFR mAb therapy in patients with mCRC has begun. Epistolio et al. (41) also reported a substantial reduction in RAS mutant cells in a majority of patients with mCRC treated with chemotherapy plus anti-VEGF mAb. There remain many questions and controversies regarding the NeoRAS-wt phenomenon. We were able to confirm that reversion to RAS-wt appears to follow a good response to first-line treatment and that RAS mutations disappear rapidly after the first cycles of chemotherapy. We also showed that use of anti-VEGF mAb is not associated with the development of NeoRAS-wt.
Our study was limited by the fact that patients’ plasma RAS status was not monitored by LB serially during the treatment period. Therefore, the exact time of reversion from RAS-mt to RAS-wt was not identified. Further, we did not check RAS status in the NeoRAS-wt group at the time we checked disease outcomes. Another limitation is the short follow-up period, which did not allow for evaluation of long-term outcomes. As noted, our study was conducted as a prospective observational study. Trials are needed to confirm the clinical meaning and the effects of anti-EGFR mAb therapy for NeoRAS-wt mCRC, including the timing of administration and whether anti-EGFR mAb treatment prolongs patients’ survival. In addition, the clinical ctDNA assay is still controversial. The American Society of Clinical Oncology and the American College of Pathologists have reported that the clinical validity and clinical utility of ctDNA assays are limited (42).
More tissue RAS-mt patients than we expected were found to be positive for plasma RAS-wt (NeoRAS-wt) during treatment for mCRC. NeoRAS-wt appears to be useful not only as a marker for a good prognosis but also as a new indicator that will expand treatment options for some patients with RAS-mt mCRC who, until now, have not been eligible for anti-EGFR mAb therapy. Further research into NeoRAS-wt is awaited to confirm this notion.
Acknowledgements
We would like to thank Prof. Tina Tajima for assistance in reviewing the manuscript to ensure proper English expression. This work was supported by JSPS KAKENHI Grant Number 19K09228.
Footnotes
Authors’ Contributions
Study conception and design: S Sato, M Shiozawa, T Oshima. Acquisition of data: S Sato, S Nukada, K Iguchi, Y Mikayama. Analysis and interpretation of data: S Sato, H Okamoto, T Kohmura, K Kazama. Manuscript writing: All authors. Critical revision and final approval of manuscript: S Sato, K Tanaka, Y Rino.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received February 20, 2022.
- Revision received March 15, 2022.
- Accepted March 16, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
