Standard first-line treatment for unresectable RAS wild-type (WT) recurrent or metastatic colorectal cancer (mCRC) includes chemotherapy combined with either an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (e.g., panitumumab or cetuximab) or an antivascular endothelial growth factor (VEGF) antibody (bevacizumab). The phase 3 PARADIGM trial demonstrated longer overall survival (OS) with first-line panitumumab plus modified 5-fluorouracil, L-leucovorin, oxaliplatin (mFOLFOX6) versus bevacizumab plus mFOLFOX6 in patients with left-sided primary tumors and in the overall population. However, exploratory analyses revealed poorer survival in patients with right-sided tumors. Differences in outcomes with anti-EGFR treatment may be attributed to tumor genomic and molecular profiles associated with primary resistance to EGFR inhibition, such as the BRAF V600E mutation and high microsatellite instability (MSI-H). Current guidelines recommend guiding anti-EGFR treatment decisions based on primary tumor location and testing for BRAF and RAS mutations, and deficient mismatch repair or MSI. Other less common alterations linked to primary resistance include mutations in PTEN and EGFR extracellular domain (ECD), amplifications of HER2 and MET, and fusions of ALK, RET, and NTRK1. While individual molecular markers have guided drug development, testing for a combination of multiple markers holds potential for more precise therapy selection. Previous studies have worked towards establishing a testing panel including a broad array of rare genomic alterations linked to primary resistance, demonstrating that detection of these alterations in tumor biopsy samples, along with primary tumor sidedness, predicts clinical outcomes on anti-EGFR therapy. Circulating tumor DNA (ctDNA) genotyping, a minimally invasive alternative to tissue biopsy, offers advantages for identifying patients who might benefit from anti-EGFR therapy. This study, a prespecified exploratory biomarker analysis of the phase 3 PARADIGM trial, tested ctDNA in baseline plasma samples from over 700 patients with RAS WT mCRC to investigate the utility of ctDNA-based negative hyperselection for predicting treatment outcomes with mFOLFOX6 combined with either panitumumab or bevacizumab.

The literature review extensively covers studies associating specific genetic alterations and right-sided tumor location with resistance to anti-EGFR treatment in mCRC. It highlights the clinical guidelines from the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) emphasizing the importance of BRAF and RAS mutation testing, and MSI status in guiding anti-EGFR treatment decisions. The review also mentions studies that have linked other less common molecular alterations, such as mutations in PTEN and EGFR extracellular domain (ECD), amplifications of HER2 and MET, and fusions of ALK, RET, and NTRK1, to primary resistance to EGFR inhibitors. The review underscores the limitations of single biomarker testing and the potential benefits of a broader panel for more precise patient selection. Several studies using tumor biopsy samples have explored the concept of negative hyperselection, demonstrating the predictive value of a comprehensive panel of genetic alterations in determining patient response to anti-EGFR therapy. Finally, the literature supports the use of ctDNA as a minimally invasive alternative to tissue biopsy for identifying potential responders to anti-EGFR treatment, highlighting the advantages of liquid biopsy over traditional tissue biopsy in this context.

This prespecified exploratory biomarker analysis of the phase 3 PARADIGM trial (NCT02394834) included 733 patients (91.4%) from the PARADIGM efficacy analysis set (n=802) who provided informed consent and had evaluable baseline plasma ctDNA samples. The ctDNA was assessed for 90 mutations, 26 amplifications, and 3 rearrangements in mCRC-related genes using a custom NGS-based panel. Negative hyperselection was defined as plasma ctDNA negative for all prespecified gene alterations associated with resistance to anti-EGFR therapy (mutations in BRAF V600E, KRAS, NRAS, PTEN, and EGFR ECD exons 1-16; amplifications of HER2 and MET; and gene fusions of RET, NTRK1, and ALK). An additional exploratory analysis assessed genetic alterations of MSS/MSI status and RAS/BRAF mutations based on guideline recommendations. Patient characteristics were summarized using descriptive statistics. The association between negative hyperselection status and OS, PFS, response rate, depth of response, and curative resection rate was evaluated. Kaplan-Meier method was used for PFS and OS analysis. Cox proportional hazard models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs), and logistic regression analysis was used to calculate odds ratios (ORs) for response rates and curative resection rates. All statistical tests were two-sided without adjustment for multiple comparisons. Analyses were performed using R (v. 4.0.5) with several statistical packages.

In the overall biomarker-evaluable population, median OS was 35.6 months with panitumumab + mFOLFOX6 and 31.6 months with bevacizumab + mFOLFOX6 (HR for death, 0.87; 95% CI, 0.73–1.02). For patients meeting negative hyperselection criteria (no gene alteration detected), OS was significantly longer with panitumumab versus bevacizumab in the overall population (40.7 vs 34.4 months; HR, 0.76; 95% CI, 0.62–0.92) and in patients with left-sided primary tumors (42.1 vs 35.5 months; HR, 0.76; 95% CI, 0.61–0.95). There was a trend towards longer OS with panitumumab in patients with right-sided tumors (38.9 vs 30.9 months; HR, 0.82; 95% CI, 0.50–1.35). In contrast, for patients with any gene alteration, median OS was similar or inferior with panitumumab versus bevacizumab, regardless of tumor sidedness. In negative hyperselected patients, PFS was similar between treatment groups in left-sided and overall populations, but shorter with panitumumab in the right-sided population. Response rates were significantly higher with panitumumab versus bevacizumab in the overall negative hyperselected population (81.5% vs 66.8%) and in the left-sided population (83.3% vs 66.5%), with a similar trend in the right-sided population. Median depth of response was greater with panitumumab in negative hyperselected patients. The curative resection rate was higher with panitumumab versus bevacizumab in negative hyperselected patients with left-sided tumors (19.8% vs 10.6%) and in the overall negative hyperselected population (18.9% vs 11.1%). When stratified by RAS/BRAF and microsatellite stability status, patients with RAS/BRAF WT and MSS/MSI-L had comparable median OS with both treatment arms, whereas patients with RAS/BRAF mutation or MSI-H showed inferior or similar OS with panitumumab compared to bevacizumab. Adverse event incidence was similar across groups.

This study demonstrates the potential prognostic and predictive value of ctDNA-based negative hyperselection for identifying patients with RAS WT mCRC who may benefit from first-line panitumumab plus chemotherapy. The findings suggest that negative hyperselection using a comprehensive ctDNA assay may be more informative for treatment selection than tumor sidedness alone, particularly for patients with left-sided tumors. The observation that even in the right-sided population, negative hyperselected patients showed numerically longer OS with panitumumab warrants further investigation. The study's comprehensive panel of gene alterations allowed the identification of a therapeutic effect by combining multiple alterations associated with resistance, even though individual alterations had low frequencies. The results support the current clinical practice of using anti-EGFR therapy for left-sided, RAS and BRAF WT, and MSS mCRC. However, the broader ctDNA-based panel improved OS prediction compared to current guideline recommendations. This suggests that expanding testing beyond current recommendations could improve patient selection for anti-EGFR therapy, particularly in the right-sided mCRC population.

This study shows that ctDNA-based negative hyperselection using a comprehensive panel of gene alterations associated with anti-EGFR resistance can identify mCRC patients who might benefit from first-line panitumumab plus chemotherapy. While tumor location remains relevant, this approach may offer improved patient selection. Further studies are needed to confirm these findings, especially regarding the benefit in right-sided mCRC.

The study has several limitations, including the lack of detailed analysis of tumor specimens, potential discrepancies between tissue and ctDNA results (due to spatial heterogeneity, timing of sampling, and assay sensitivity differences), challenges in detecting gene fusions and amplifications in ctDNA, and the lack of statistical power for comparisons between specific subgroups. Further investigation is needed to address these limitations and validate the findings.