
==== Front
JCO Precis Oncol
JCO Precis Oncol
po
PO
JCO Precision Oncology
2473-4284
Wolters Kluwer Health

39178370
PO.24.00031
10.1200/PO.24.00031
00196
ORIGINAL REPORTS
Targeted Drug Therapy
Kinetic Profiling of RAS Mutations With Circulating Tumor DNA in the Canadian Cancer Trials Group CO.26 Trial Suggests the Loss of RAS Mutations in Neo-RAS-Wildtype Metastatic Colorectal Cancer Is Transient
https://orcid.org/0000-0002-6439-4606
Wu Florence T.H. MD, PhD 1
Topham James T. MSc 1
O'Callaghan Chris J. PhD, DVM, MSc 2
https://orcid.org/0000-0002-1388-2183
Feilotter Harriet PhD 2
https://orcid.org/0000-0002-3211-0656
Kennecke Hagen F. MD, MHA, FRCPC 3
Drusbosky Leylah PhD 4
Renouf Daniel J. MD, FRCPC 1
Jonker Derek J. MD, FRCPC 5
https://orcid.org/0000-0003-4842-2184
Tu Dongsheng PhD 2
https://orcid.org/0000-0002-4581-8848
Chen Eric X. MD, PhD, FRCPC 6
https://orcid.org/0000-0001-8189-2132
Loree Jonathan M. MD, MS 1
1 BC Cancer, University of British Columbia, Vancouver, BC, Canada
2 Canadian Cancer Trials Group, Kingston, ON, Canada
3 Oregon Health & Science University, Portland, OR
4 Guardant Health, Redwood City, CA
5 The Ottawa Hospital Research Institute, University of Ottawa, Ottawa, ON, Canada
6 Princess Margaret Cancer Centre, Toronto, ON, Canada
Jonathan M. Loree, MD, MS; Twitter: @jonathanloree; e-mail: jonathan.loree@bccancer.bc.ca.
2024
23 8 2024
23 8 2024
8 e240003119 1 2024
18 6 2024
26 7 2024
© 2024 by American Society of Clinical Oncology
2024
American Society of Clinical Oncology
https://creativecommons.org/licenses/by-nc-nd/4.0/ Creative Commons Attribution Non-Commercial No Derivatives 4.0 License: http://creativecommons.org/licenses/by-nc-nd/4.0/

PURPOSE

In metastatic colorectal cancer (mCRC), RAS mutations drive resistance to anti–epidermal growth factor receptor antibodies. It is unclear whether RAS mutations ever become clonally undetectable.

METHODS

CO.26 was a phase II clinical trial that assessed durvalumab + tremelimumab in heavily pretreated mCRC. RAS mutation status was tracked over time using circulating tumor DNA (ctDNA) sequencing at baseline, week 8, and on progression.

RESULTS

Among the 95 patients with KRAS/NRAS mutations in their archival tumor tissue, 6.3% (6/95) had undetectable RAS mutations in ctDNA collected at baseline or week 8 of the CO.26 study. Of these, 67% (4/6) of disappearances were transient, with the same mutation reappearing with progressive disease. In three cases, the simultaneous persistence of other preexisting CRC-associated truncal mutations could not be demonstrated, suggestive of low tumor shedding of ctDNA, leaving the incidence of true clonal reversion to RAS-wildtype (WT) possibly as low as 3.2% (3/95). Fewer patients in the neo-RAS-WT group (33%) had greater than four lesions at trial baseline compared with patients with persistent RAS mutations (75%), P = .046. The likelihood of synchronous metastases at cancer diagnosis (33% v 63%; P = .15) or liver metastases at trial baseline (50% v 68.5%; P = .17) was not significantly different between patients with disappearing versus persistent RAS mutations. Overall survival from stage IV diagnosis (hazard ratio, 0.77 [95% CI, 0.35 to 1.72]; P = .52) was not significantly different between those with disappearing versus persistent RAS mutations. The disappearance of RAS mutations was not associated with primary tumor sidedness (P = .41), archival BRAF/MEK/ERK-mutant status (P = .16/1.00/.09), nor baseline ctDNA HER2 amplifications (P = 1.00).

CONCLUSION

We identified a 3.2%-6.3% prevalence of the neo-RAS-WT phenomenon in the CO.26 trial. However, 67% of apparent cases were transient with subsequent re-emergence.

Serial ctDNA shows transience of neo-RAS-wildtype phenomenon in mCRC [CCTG CO26].

OPEN-ACCESSTRUE
==== Body
pmcINTRODUCTION

Anti–epidermal growth factor receptor (EGFR) monoclonal antibodies, such as cetuximab or panitumumab, are effective therapies in KRAS/NRAS (RAS)-wildtype (WT) metastatic colorectal cancer (mCRC).1 However, RAS mutations occurring in approximately 55% of mCRCs preclude benefit from anti-EGFR therapy.1 Even when RAS-WT disease initially responds to anti-EGFR therapy, secondary resistance often develops through acquisition of RAS or EGFR ectodomain mutations.1-5 With the incorporation of circulating tumor DNA (ctDNA) into clinical practice, it has now become possible to track mutation profiles serially via liquid biopsies. RAS-mutant disease may evolve over time to lose intrinsic or acquired RAS mutations, potentially rendering these cancers newly susceptible to anti-EGFR therapies.6,7

CONTEXT

Key Objective

Neo-RAS-wildtype (WT) is increasingly used to describe cases of metastatic colorectal cancer (mCRC) where intrinsic KRAS/NRAS mutations disappear over time. This terminology remains controversial since most studies have not demonstrated persistently undetectable RAS mutations in circulating tumor DNA (ctDNA) against a backdrop of other retained CRC-associated truncal mutations. In this study, we assessed the durability of disappearing RAS mutations and the frequency of simultaneous persistence of CRC-associated truncal mutations.

Knowledge Generated

Serial ctDNA analysis from the Canadian Cancer Trials Group CO.26 trial show a 6.3% prevalence of the neo-RAS-WT phenomenon, 67% (4/6) of which were transient, with the same initial RAS mutations reappearing within a year. In 50% (3/6) of the neo-RAS-WT cases, the persistence of CRC-associated truncal mutations could not be confirmed.

Relevance

Our study suggests that low tumor fraction and ctDNA sensitivity are potential confounders that need to be considered in clinical trials of anti–epidermal growth factor receptor therapy in the neo-RAS-WT mCRC population.

Disappearing RAS mutations has been referred to as neo-RAS-WT in recent literature; several small studies have estimated its prevalence to be in the range of 6.6%-67%.8-14 A larger longitudinal study of 464 patients from three cohorts provided more conservative estimates of 1.6%-8.8%.15 These findings have prompted phase II trials (MoLiMoR, KAIROS, ConVertix, CETIDYL, and C-PROWESS) that are testing anti-EGFR therapy after detection of a neo-RAS-WT phenotype.16,17 Success of these trials depends on the durability and inherent prognostic value of the neo-RAS-WT phenomenon—both unanswered questions on the basis of current literature. Cases that are durable through serial sampling should theoretically be more responsive to anti-EGFR therapy.

To address these knowledge gaps, we analyzed neo-RAS-WT cases from CO.26, an open-label randomized phase II multicenter clinical trial conducted by the Canadian Cancer Trials Group (CCTG).18 Most patients had retrievable archival tumor tissue collected at the time of initial CRC diagnosis. Baseline and serial plasma for ctDNA analysis were collected before and during treatments while on the CO.26 trial (ClinicalTrials.gov identifier: NCT02870920), creating an opportunity to assess the stability of RAS mutations between archival tissue and subsequent time points over the course of the trial.

METHODS

Patient Population

CO.26 (ClinicalTrials.gov identifier: NCT02870920) was an unblinded phase II trial that randomly assigned 180 patients with treatment-refractory mCRC 2:1 to receive durvalumab (a fully human monoclonal IgG1κ antibody against PD-L1; given as 1,500 mg intravenously once every 4 weeks) plus tremelimumab (a fully human monoclonal IgG2 antibody against CTLA-4; given as 75 mg intravenously once every 4 weeks for the initial four cycles only), versus best supportive care alone, after written and informed consent (Fig 1). Patients had received all available standard therapies before enrollment. The study received institutional review board approval and was conducted per the Declaration of Helsinki and International Ethical Guidelines for Biomedical Research Involving Human Subjects. Full study details and the study protocol are available as previously described.18

FIG 1. CONSORT diagram for CCTG CO.26 study adapted from Chen et al.18 Post hoc analysis of circulating tumor DNA highlighted in gray. BSC, best supportive care; CCTG, Canadian Cancer Trials Group; cfDNA, cell-free DNA; DT, durvalumab plus tremelimumab.

ctDNA Analyses

The CO.26 study collected plasma for ctDNA analysis at study entry (baseline), 8 weeks after initiating study therapies, and upon disease progression.18 Plasma was assessed through next-generation sequencing using the Guardant OMNI panel (2.15 Mb, 500-gene panel, Guardant Health, Inc, Palo Alto, CA) to identify single-nucleotide variants (SNVs), indels (insertion/deletions), fusions, and copy-number variations (CNVs), as well as determine microsatellite instability status and tumor mutational burden.18 The Guardant OMNI panel was previously validated as having 98.7% accuracy at detecting SNVs with 95% limits of detection of 0.24%-0.6% variant allele fraction (VAF).19

Tissue-Based Analyses

Archival formalin-fixed and paraffin-embedded (FFPE) tumor tissue and leukocytes from peripheral blood were used for tumor and matched normal sequencing, respectively. The Allprep DNA/RNA FFPE kit was used to purify DNA and total RNA per manufacturer's directions from tissue, and the QIAamp DNA Minikit was used for DNA extraction from blood-derived leukocytes. Sequencing libraries were created using the Agilent SureSelectXT method with subsequent hybridization and capture with SureSelect Human All Exon v6 baits (Agilent Technologies, Santa Clara, CA) for whole-exome sequencing (WES). Quantified, normalized, and pooled libraries were multiplexed and sequenced using an Illumina platform with a sequencing protocol of 100 bp paired-end sequencing, with a read depth necessary to reach and average coverage before deduplication of 100× for normal or 400× for tumor samples. Somatic variants were called on tumor-normal pairs through a combination of Manta v1.5.020 and Strelka v2.9.10,21 using default parameters and genome build GRCh37/hg19. Variants were annotated using SnpEff v4.322 with parameters -v GRCh37.75 -canon -no-downstream -no-upstream -noLog -noStats -no-intergenic. variant call format files were converted to mutation annotation format using vcf2maf v1.6.18 with default parameters. Variants were filtered to exclude those located outside of exons (using consensus exon regions GRCh37.p13; GCF_000001405.25, downloaded June 19, 2020) and any common variants identified by ExAC23 (ExAC_nonTCGA.r0.3.1.sites.vep.vcf.gz). Variants were further filtered on the basis of established guidelines,24,25 including VAF ≥0.05, tumor depth ≥25, and alternate allele count ≥3. Only missense, nonsense, and in-frame/frameshift somatic variants were included for analysis. For the 59 patients with no tissue for WES, we referred to clinical entries in the trial database for original KRAS/NRAS mutational status as determined by institution-specific companion diagnostic platforms looking at known hotspots.

Variant Allele Fraction

VAF was defined as the number of covering reads containing a given variant allele at a given position divided by the total number of reads overlapping that position. For a given variant, the relative VAF (rVAF) was defined as the VAF of that variant divided by the maximum VAF of any somatic variant in the same patient sample.

Statistical Analysis

GraphPad Prism (San Diego, CA) and R v3.6.3 were used for statistical analysis. Continuous data were subjected to Mann-Whitney tests (two-sampled) or Kruskal-Wallis tests (with Dunn's post-tests on planned comparisons) for non-Gaussian distributions; and t-tests (two-sampled) or one-way analysis of variance (with Bonferroni multiple test correction) for Gaussian distributions. Binomial proportions were subjected to Fisher's exact tests or chi-squared tests as appropriate. Kaplan-Meier survival curves were assessed via log-rank tests.

RESULTS

Stability of RAS Mutational Status Over Time

The 169 patients in the CO.26 trial with ctDNA sequencing available were divided into four categories on the basis of whether the KRAS and NRAS mutation status (WT v mutated) of their CRC changed between time points (Fig 2). Of the 74 patients initially diagnosed with RAS-WT mCRC, 30 remained RAS-WT throughout serial ctDNA (group A; persistent WT). The remaining 44 patients acquired KRAS/NRAS mutations by the time of baseline (98%) or week-8 (2%) ctDNA sampling (group B; acquired mutant). Of these, 22 patients acquired KRAS mutations, four acquired NRAS mutations, while 18 acquired both KRAS and NRAS mutations.

FIG 2. CCTG CO.26 participants grouped by stability of KRAS and NRAS mutation status over time. Thirty patients were in group A with RASWT→ RASWT (persistent WT). Forty-four patients were in group B with RASWT→RASmut (acquired mut). Eighty-nine patients were in group C with RASmut→RASmut (persistent mutant), and six patients were in group D with RASmut→RASWT (neo-RAS-WT). Inner rings depict patient KRAS and NRAS mutational status over four time points. Outer barplots illustrate the baseline TMB score and primary tumor location of each patient sample. CCTG, Canadian Cancer Trials Group; mut, mutant; NA, not applicable; TMB, tumor mutational burden; WT, wildtype.

Of the 95 patients initially diagnosed with RAS-mutated mCRC, 89 (93%) retained their KRAS or NRAS mutation over time (group C; persistent mutant). For the remaining six patients (6.3%), the initial RAS mutation became undetectable by the time of baseline (83%) or week-8 (17%) ctDNA sampling (group D: neo-RAS-WT cases). All six patients had initially KRAS-mutated NRAS-WT disease—specifically, two had a KRAS G12D mutation (by WES), another two had a KRAS G12V mutation (by WES), one had a KRAS A146T mutation (by WES), and one had a KRAS mutation without the reported specific mutated codon in the trial database (by clinical assay) in their archival sample.

Durability of Neo-RAS-WT Cases and Persistence of Truncal Mutations

For the six neo-RAS-WT cases, the rVAF of their KRAS mutations were plotted over time (Fig 3). The loss of RAS mutations was often transient. Four patients reacquired KRAS-mutant status either by week 8 or by the time of progressive disease (PD), with return of the same variant between 55 and 293 days (median, 162.5 days) after baseline. There were only two potentially durable cases. One patient had a sustained neo-RAS-WT status that could be confirmed at PD (174 days from baseline; patient 169 in Fig 3D). Another patient had sustained neo-RAS-WT status at least up to week 8 but did not have a plasma ctDNA sample at PD to confirm end point stability (patient 166 in Fig 3C).

FIG 3. Clonal variant dynamics during neo-RAS-WT reversions. These six patients had colorectal cancer that was initially KRAS-mutated, which then became KRAS-WT over time. In each panel, the top three most clonal variants (defined by highest rVAF) are depicted for each time point along with any variants meeting this criterion in previous time points for that sample. Percent next to gene names indicate the rVAF of that variant at the first time point it was observed for that sample. Synonymous mutations are not shown in the figure but were used to calculate rVAF. rVAF, relative variant allele fraction; WT, wildtype.

In three of the six cases, at least one CRC-associated truncal mutation was detectable by ctDNA at the time of neo-RAS-WT phenomenon (eg, ATM for patients 167 and 169 in Figs 3B and 3D; TP53 for patient 166 in Fig 3C). In the remaining cases, the persistence of other CRC-associated truncal alterations could not be confirmed at the time RAS mutations were lost (eg, TP53 and SMAD4 mutations disappeared at week 8 for patient 168 in Fig 3A; TP53 mutation disappeared at CO.26 baseline for patient 165 in Fig 3E). These observations suggest that at least 50% (3/6) of our transient cases of neo-RAS-WT phenomenon may be confounded by limitations in ctDNA sensitivity. Of note, the mean VAF was significantly lower in neo-RAS-WT cases compared with all other cases at all three time points of ctDNA analysis (Fig 4): at baseline (P = .0011), week 8 (P = .0018), and on progression (P = .021). These differences may reflect lower tumor burden or shedding in neo-RAS-WT cases, although other tumor-related factors (eg, rates of proliferation and necrosis; degree of tumor vascularization) or host-related factors (eg, organ sites of disease) may contribute as well.26

FIG 4. Mean VAF in neo-RAS-WT cases versus all other cases across each ctDNA time point. Neo-RAS-WT cases (n = 5) showed significantly (P < .05) lower mean VAF levels (all variants included) compared with all other cases at baseline (P = .0011), week 8 (P = .0018), and on progression (P = .021). Median, 25%-75% IQR, and Wilcoxon mean rank-sum P values are shown. ctDNA, circulating tumor DNA; VAF, variant allele fraction; WT, wildtype.

Neo-RAS-WT Phenotype Is Not Prognostic for Overall Survival

Overall survival (OS), defined as the time from date of initial cancer diagnosis to death, was not statistically significantly different between the neo-RAS-WT group (median OS of 7.7 years, n = 6), persistent RAS-WT group (4.5 years, n = 30), acquired RAS-mutant group (4.8 years, n = 44), and persistent RAS-mutant group (4.2 years, n = 89), with P = .44 by log-rank test (Fig 5A). Variations in sequence or specific agents used in previous lines of therapy in this heavily pretreated population (summarized in Table 1) may have obscured potential OS differences. The proportion of patients with synchronous metastatic disease at the time of cancer diagnosis was not statistically different between the neo-RAS-WT group versus the other three groups combined (33% v 59%; P = .40). OS, redefined as the time from stage IV disease to death, was also not statistically significantly different between the neo-RAS-WT group (median OS of 3.9 years, n = 6), persistent RAS-WT group (2.6 years, n = 30), acquired RAS-mutant group (4.2 years, n = 44), and persistent RAS-mutant group (3.2 years, n = 89), with P = .20 by log-rank test (Fig 5B). The duration between cancer diagnosis and random assignment into the CO.26 trial was not statistically significantly different between the neo-RAS-WT group (median, 5.6; range, 0.8-8.0 years), the persistent RAS-WT group (median, 3.6; range, 1.5-12.7 years), acquired RAS-mutant group (median, 3.4; range, 0.6-15.1 years), and persistent RAS-mutant group (median, 3.4; range, 0.6-15.1 years), P = .65. In terms of baseline characteristics upon enrollment onto CO.26, fewer neo-RAS-WT cases had greater than four lesions compared with the other three groups combined (33% v 74%; P = .03), but there were no statistically significant differences in terms of presence of liver metastases (50% v 71%; P = .26) or larger-than-10-cm lesions (0% v 10%; P = .42; Table 1).

FIG 5. Kaplan-Meier analyses of OS. (A) OS as defined from initial cancer diagnosis to death. Of note, the neo-RAS-WT group had lower rates of synchronous metastatic disease at initial diagnosis than others. (B) OS as defined from stage IV diagnosis to death. HR, hazard ratio; mut, mutant; OS, overall survival; WT, wildtype.

TABLE 1. Categorization of 169 Patients From the CO.26 Trial Into Four Groups on the Basis of Evolution of KRAS and NRAS Mutation Status

Treatment Characteristic	Group A Persistent RAS-WT (n = 30)	Group B Acquired RAS-Mutant (n = 44)	Group C Persistent RAS-Mutant (n = 89)	Group D Neo-RAS-WT (n = 6)	P	
Groups A to D	Groups C v D	
 Sex, No. (%)					.19	1.00	
  Female	10 (33.3)	9 (20.5)	35 (39.3)	2 (33.3)			
  Male	20 (66.7)	35 (79.5)	54 (60.7)	4 (66.7)			
 Race, No. (%)							
  White	25 (83.3)	39 (88.6)	74 (8,310)	5 (83.3)	.76	1.00	
  Black	0	2 (4.5)	1 (1.1)	0			
  Native Hawaiian	1 (3.3)	0	0	0			
  Asian	4 (13.3)	2 (4.5)	11 (12.4)	1 (16.7)			
  Indigenous	0	1 (2.3)	2 (2.2)	0			
  Not reported	0	0	2 (2.2)	0			
  Unknown	0	0	1 (1.1)	0			
 Age, years, median (min-max)	68 (48-87)	65 (46-85)	65 (39-85)	63 (44-78)			
 Age ≥65, years, No. (%)	18 (60.0)	23 (52.3)	47 (52.8)	3 (50.0)	.90	1.00	
 ECOG performance status, No. (%)					.08	0.39	
  0	3 (10.0)	13 (29.5)	28 (31.5)	3 (50.0)			
  1	27 (90.0)	31 (70.5)	61 (68.5)	3 (50.0)			
 Months from first histologic diagnosis to random assignment, median (min-max)	46 (14-157)	43 (17-153)	41 (8-181)	67 (10-95)	.65	0.51	
 Presence of liver metastases, No. (%)	19 (63.3)	36 (81.8)	61 (68.5)	3 (50.0)	.19	0.39	
 Sidedness, No. (%)					.03	0.54	
  Left	22 (73)	40 (91)	54 (61)	5 (83)			
  Right	8 (27)	4 (9)	34 (38)	1 (17)			
  Unknown	0	0	1 (1)	0			
 Treatment, No. (%)					.053	0.13	
  Durvalumab + tremelimumab	17 (56.7)	28 (63.6)	70 (78.7)	3 (50.0)			
  Best supportive care	13 (43.3)	16 (36.4)	19 (21.3)	3 (50.0)			
Specific previous chemotherapy, No. (%)							
 Previous thymidylate synthase inhibitor	30 (100.0)	44 (100.0)	89 (100.0)	6 (100.0)	NA	NA	
 Previous irinotecan-containing regimen	29 (96.7)	43 (97.7)	89 (100.0)	4 (66.7)	<.0001	0.003	
 Previous oxaliplatin-containing regimen	23 (76.7)	40 (90.9)	76 (85.4)	5 (83.3)	.41	1.00	
 Previous cetuximab or panitumumab	26 (86.7)	32 (72.7)	7 (7.9)	0	<.0001	1.00	
 Previous VEGF-targeting therapy	22 (73.3)	41 (93.2)	78 (87.6)	3 (50.0)	.008	0.04	
  Bevacizumab	18 (60.0)	39 (88.6)	74 (83.1)	2 (33.3)			
  Regorafenib	11 (36.7)	13 (29.5)	20 (22.5)	1 (16.7)			
 Previous TAS-102 therapy	0	0	0	0	NA	NA	
Extent of disease (target and nontarget lesions)							
 No. of target and nontarget lesions							
  Median (min-max)	6 (3-11)	6 (3-12)	6 (1-13)	4 (2-7)			
   1, No. (%)	0	0	2 (2.2)	0			
   2, No. (%)	0	0	2 (2.2)	2 (33.3)			
   3, No. (%)	4 (13.3)	8 (18.2)	9 (10.1)	0			
   4, No. (%)	3 (10.0)	5 (11.4)	9 (10.1)	2 (33.3)			
   ≥5, No. (%)	23 (76.7)	31 (70.5)	67 (75.3)	2 (33.3)	.15	.046	
 Largest target lesion in cm							
  Median (min-max)	5 (2-14)	7 (2-15)	5 (2-19)	5 (2-10)			
  <2, No. (%)	1 (3.4)	1 (2.3)	7 (7.9)	1 (16.7)			
  2-5, No. (%)	14 (48.3)	13 (29.5)	41 (46.1)	2 (33.3)			
  >5-10, No. (%)	10 (34.5)	25 (56.8)	32 (36.0)	2 (33.3)			
  >10, No. (%)	4 (13.8)	5 (11.4)	7 (7.9)	0	.66	1.00	
Abbreviations: ECOG, Eastern Cooperative Oncology Group; max, maximum; min, minimum; NA, not applicable; VEGF, vascular endothelial growth factor; WT, wildtype.

Neo-RAS-WT Phenomenon Did Not Correlate With Clinically Relevant Tumor Characteristics

Among patients initially diagnosed with RAS-mutated CRC, there was no significant association between left-/right-sidedness of primary tumors and neo-RAS-WT status (8% v 3%; P = .41; Table 1). The neo-RAS-WT phenomenon was not significantly associated with the presence of BRAF/MEK/ERK mutations in archival tissue (P > .05; Data Supplement, Table S1), nor with BRAF/MEK/ERK SNVs or HER2 CNVs in baseline plasma ctDNA (P > .05; Data Supplement, Table S1).

DISCUSSION

In the CO.26 study, the neo-RAS-WT phenomenon was observed in 6.3% (6/95) of patients who had RAS-mutated disease at the time of initial CRC diagnosis. This rate is consistent with the 1.6% to 8.8% range that has previously been reported.15 Uniquely, our study incorporated serial ctDNA sequencing that enabled assessment of the kinetics and stability of the neo-RAS-WT phenotypes over time. Six neo-RAS-WT cases were detected within 8 weeks of trial enrollment, but at least four of these were only transient with the same KRAS-mutant variant re-emerging by the time of disease progression. This lack of durability in 67% of cases raises the question of whether the neo-RAS-WT phenomenon can be a clinically meaningful therapeutic target. Currently, at least five single-arm phase II clinical trials are underway looking at the potential clinical significance of neo-RAS-WT cases.16,17 A study by Raimondi et al10 has hinted at the potential transient nature of the neo-RAS-WT phenomenon, reporting the reappearance of RAS mutations after neo-RAS-WT tumors were exposed to anti-EGFR therapy.

We observed instances of simultaneous disappearance of other CRC-associated truncal mutations at the same time when RAS mutations were lost, followed by their concurrent re-emergence. We estimated an up to 50% false-negative rate in our study, with three of six apparent neo-RAS-WT cases potentially resulting from transient states of low ctDNA shedding. To our knowledge, none of the neo-RAS-WT studies in the published literature have sought to demonstrate a persistent strong presence of CRC-specific truncal mutations as a positive control for ongoing detectability of tumor content. One study used persistent levels of WIF1-promotor CpG island hypermethylation, which authors pointed out is an epigenetic alteration present in 70%-90% of CRC, as a positive control for eight of the 11 reported neo-RAS-WT cases.8 However, WIF1-promotor hypermethylation is seen in a variety of other tumor types as well.27-29 Until the field adopts more stringent controls, it will remain difficult to discern the fraction of reported cases of neo-RAS-WT phenomena that truly stem from tumor clonal dynamics in action, whereby the original RAS-mutant tumor clone becomes transiently or durably masked by other clones that rise to dominance under selective pressures.

Our study is limited by the post hoc nature of our neo-RAS-WT analysis. As such, the CO.26 trial was not designed to mandate rebiopsies of tumor tissue at the time of ctDNA detection of neo-RAS-WT cases to allow for definitive contemporaneous confirmation of genotypic reversion. Another limitation stems from the fact that some patients did not have a complete set of evaluable data available from each time point (archival, baseline, week 8, and on progression), thus restricting our ability to fully track variant kinetics over time.

In conclusion, this study presents ctDNA-based estimation of the rare neo-RAS-WT phenomenon. Our data suggest that the majority of neo-RAS-WT cases are transient rather than durable. This has significant implications for anti-EGFR efficacy being evaluated in neo-RAS-WT CRCs and suggests serial tissue–based testing should be considered to help confirm that RAS WT status is not confounded by limitations in ctDNA sensitivity.

PRIOR PRESENTATION

SUPPORT

DATA SHARING STATEMENT

A data sharing statement provided by the authors is available with this article at DOI https://doi.org/10.1200/PO.24.00031.

AUTHOR CONTRIBUTIONS

Conception and design: Harriet Feilotter, Hagen F. Kennecke, Eric X. Chen

Provision of study materials or patients: Hagen F. Kennecke, Daniel J. Renouf, Derek J. Jonker, Eric X. Chen

Collection and assembly of data: Leylah Drusbosky, Derek J. Jonker, Dongsheng Tu, Eric X. Chen

Data analysis and interpretation: Florence T.H. Wu, James T. Topham, Hagen F. Kennecke, Daniel J. Renouf, Derek J. Jonker, Dongsheng Tu, Eric X. Chen, Jonathan M. Loree

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated unless otherwise noted. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

Open Payments is a public database containing information reported by companies about payments made to US-licensed physicians (Open Payments).

Presented at 2023 ASCO Annual Meeting (poster presentation 3567).

Supported by the Canadian Cancer Society. J.M.L. and D.J.R. have received Michael Smith Health Professional Investigators awards, which helped support this study. Funds from the BC Cancer Foundation helped support this study. AstraZeneca provided durvalumab and tremelimumab as well as financially supported sequencing costs associated with correlative analyses.

Florence T.H. Wu

Honoraria: Bristol Myers Squibb

Harriet Feilotter

Honoraria: Roche Canada, Precision RxDx, AstraZeneca

Research Funding: AstraZeneca (Inst), Lilly (Inst), EMD Serono (Inst), Amgen (Inst), Roche Canada (Inst)

Hagen F. Kennecke

Honoraria: Natera, Exelixis

Speakers' Bureau: Natera

Research Funding: Taiho Pharmaceutical (Inst), Novartis (Inst), Exelixis (Inst)

Leylah Drusbosky

Employment: Guardant Health

Stock and Other Ownership Interests: Guardant Health

Travel, Accommodations, Expenses: Guardant Health

Daniel J. Renouf

Honoraria: Roche, Bayer, Ipsen

Consulting or Advisory Role: Roche, Bayer, Viatris

Research Funding: Bayer, Roche (Inst)

Derek J. Jonker

Consulting or Advisory Role: AstraZeneca

Eric X. Chen

Honoraria: Eisai, Bayer, AstraZeneca Canada, Ipsen, Pfizer, Merck, Roche, GlaxoSmithKline

Research Funding: Merck Sharp & Dohme, AstraZeneca/MedImmune, Bristol Myers Squibb, Novartis, Mirati Therapeutics, Roche Canada, 1Globe Health Institute, Repare Therapeutics, Nubiyota

Jonathan M. Loree

Consulting or Advisory Role: Taiho Pharmaceutical, Ipsen, Bayer, Amgen, Pfizer, Advanced Accelerator Applications, Merck, SAGA Diagnostics, Guardant Health

Research Funding: Ipsen (Inst), Amgen (Inst), Personalis

No other potential conflicts of interest were reported.
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