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Utility of Serial ctDNA in Guiding Anti-EGFR Rechallenge in Metastatic Colorectal Cancer

Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions that frequently co-occur as subclonal alterations
07 Sep 2022
Anticancer agents & Biologic therapy;  Personalised medicine

Analysis of ctDNA used with paired exome sequencing from archival tissues of patients with RAS wild-type metastatic colorectal cancer (mCRC) found that acquired resistance to anti-EGFR antibodies occurs via a multiplicity of concurrent subclonal alterations, with 21% of patients who had ≥ 10 putative concurrent resistance mechanisms. The study team also uncovered significantly more copy number amplifications in patients with anti-EGFR exposure compared with those without anti-EGFR exposure (82% versus 49%). Similarly, fusions were found to occur in 15% of patients treated with anti-EGFR antibodies compared with 1% of patients without prior anti-EGFR treatment. Serial ctDNA samples showed that while most acquired single nucleotide variants (SNVs) and fusions decay, copy number variants appeared stable over time. Patients with prior anti-EGFR treatments also had an increase in relative tumour mutation burden (TMB) and although acquired SNVs decayed over time, this induction of TMB remained stable. The findings are published by Dr. Jonathan M. Loree of the University of British Columbia in Vancouver, BC, Canada and colleagues on 25 August 2022 in the Journal of Clinical Oncology.

Primary tumour location and presence of mutations in KRAS, NRAS, and BRAF are important considerations for treatment with anti–EGFR antibodies in mCRC. Tumours with mutations in these genes do not respond to anti-EGFR antibodies and patients with right-sided tumours derive less benefit from these drugs. However, patients eventually develop resistance to anti-EGFR antibodies, with acquired KRAS, NRAS, BRAF, and EGFR ectodomain mutations accounting for approximately 50% of acquired resistance and less common alterations, such as gene fusions, also being reported.

The authors wrote in the background that longitudinal ctDNA studies have shown that among patients with acquired mutations, resistant clones decay with time, resulting in an opportunity for anti-EGFR rechallenge.  In the CRICKET and CHRONOS studies, patients previously treated with chemotherapy plus anti-EGFR antibodies were rechallenged with cetuximab plus irinotecan or panitumumab and demonstrated meaningful progression-free survival with retreatment.  Furthermore, the CAVE study demonstrated that cetuximab plus avelumab can be an active and well-tolerated rechallenge strategy in patients with pretreated RAS wild-type mCRC.

In total, 169 patients with treatment-refractory mCRC enrolled on the CO.26 study had pre–anti-EGFR tissue whole-exome sequencing compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between 66 patients with prior anti-EGFR treatment and those without. Anti-EGFR treatment occurred a median of 111 days before ctDNA assessment. The study team interrogated matched pretreatment tissue and serial ctDNA from the CO.26 study, which compared durvalumab plus tremelimumab versus best supportive care in treatment-refractory mCRC to identify novel mechanisms of resistance and describe their kinetics. At the time of recruitment to CO.26, anti-EGFR antibodies were used almost exclusively in the third-line setting in Canada, and there was limited access to regorafenib or trifluridine/tipiracil.

ctDNA identified 12 genes with increased mutation frequency after anti-EGFR treatment, including EGFR (p = 0.0007), KRAS (p = 0.0017), LRP1B (p = 0.0046), ZNF217 (p = 0.0086), MAP2K1 (p = 0.018), PIK3CG (p = 0.018), BRAF (p = 0.048), and NRAS (p = 0.048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR (p < 0.0001), SMO (p < 0.0001), BRAF (p < 0.0001), MET (p = 0.0002), FLT3 (p = 0.0002), NOTCH4 (p = 0.0006), ERBB2 (p = 0.004), and FGFR1 (p = 0.006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR treatment.

Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR treatment compared with 5% in those without (p = 0.010). Although TMB did not differ prior treatment (p = 0.63), anti-EGFR exposure increased TMB (p = 0.028), whereas lack of anti-EGFR exposure resulted in declining TMB (p = 0.014).

This study should be interpreted in the context of several limitations. However, despite the limitations, the authors commented that it presents one of the most comprehensive overviews of anti-EGFR resistance mechanisms to date. It provides serial identification and tracking of resistance patterns using a ctDNA panel assaying 500 genes, surpassing the genomic breadth of previous studies. The study supports the utility of serial ctDNA in guiding anti-EGFR rechallenge.

Improved understanding of acquired resistance may improve mCRC outcomes by informing ctDNA panels that can track tumour kinetics and monitor for resistance before radiographic progression, identifying combination strategies with anti-EGFR antibodies to prevent the development of resistance, and improving patient selection for anti-EGFR rechallenge by identifying non-RAS/BRAF/EGFR alterations that preclude benefit. Awareness of the mechanisms identified will facilitate improved tracking of and potential combinatorial strategies to delay or overcome resistance.

This work was supported by philanthropic donations received through the BC Cancer Foundation and funding provided by the Terry Fox Research Institute. Costs associated with running the CO.26 study and sequencing were supported by AstraZeneca. The Canadian Cancer Trials Group by the Canadian Cancer Society. Two authors are recipients of the MSFHR Health Professional-Investigator Award.


Topham JT, O’Callaghan CJ, Feilotter H, et al. Circulating Tumor DNA Identifies Diverse Landscape of Acquired Resistance to Anti–Epidermal Growth Factor Receptor Therapy in Metastatic Colorectal Cancer. JCO; Published online 25 August 2022. DOI: 10.1200/JCO.22.00364

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