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SDIRSACR Oncology Insights

was collected in anticoagulant tubes (EDTA/PAXgene tubes), from which plasma was separated by centrifugation at
2000g at 4 C for 10 minutes, according to the protocol. Circulating free DNA (cfDNA) was extracted using the QIAamp
o
MinElute ccfDNA Mini Kit (Qiagen, Hilden, Germany) from a plasma volume of 4ml, and the concentration of cfDNA was
measured using the Qubit™ 4 Fluorometer with the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham,
MA, USA). The extracted samples were subjected to mutation testing using the KRAS Mutation Test v2 (LSR) and/
or BRAF/NRAS Mutation Test (LSR) (Roche Diagnostics, Mannheim, Germany), a qPCR-based method designed to
detect somatic mutations in the KRAS, NRAS, and BRAF genes. Both tests are designed to include detection from DNA
extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue and blood. The KRAS Mutation Test v2 was designed to
detect the following mutations: G12X and G13X in exon 2; A59X and Q61X in exon 3; A117X and A146X in exon 3. The
BRAF/NRAS Mutation Test was intended to detect NRAS mutations in the same codons as well as in the KRAS gene, and
also BRAF mutations in codons 469 (exon 11) and 600 (exon 15).
Results: The concentration of cfDNA was from 0.106 to 29 ng/µl. Variant allele frequency (VAF) values ranged from
10.5 to 23.5. In patients who had received cetuximab, 12 (80%) had at least one mutation, whereas 3 (20%) had no
mutations in any examined genes. In the group of patients who had received panitumumab, 6 (60%) showed one or
two mutations, whereas 4 (40%) did not harbor mutations in any of the examined genes. When considering both drugs,
a total of 27 mutations in the KRAS and NRAS genes mutations were detected, in the following order for KRAS gene:
8 (29.63%) in codon 12, 7 (25.93%) in codon 61, 2 (7.41%) in codon 13, 1 (3.7%) in the codon 59, and also 1 (3.7%)
in the codon 146. Also, for the NRAS gene, detected mutations were as follows: 6 (22.22%) in codon 13, 1 (3.7%) in
codon 12, and 1 (3.7%) in codon 61. All wt KRAS patients were subsequently tested for the presence of NRAS and BRAF
mutations. Of the 15 patients who received cetuximab, 8 (53.33%) showed at least one mutation in exon 2,3, or 4 in the
KRAS gene. For four patients with detected KRAS mutations, a further analysis was performed for BRAF/NRAS genes to
examine tumor heterogeneity. Only in one patient, who received cetuximab, four concurrent KRAS mutations (G12X,
A59X, A146X, Q61X) were detected, three mutations in NRAS (G12X, G13X, Q61X), as well as a mutation in exon 11
of the BRAF gene. This was the only patient in the entire cohort with a confirmed BRAF mutation, accounting for 4%
mutated BRAF of the total of 25 patients. Overall, when considering both drugs, as well as the presence of mutations
in both genes, 18 (72%) patients had at least one mutation, while in 7 (28%) patients, no mutation was detected in the
KRAS/NRAS/BRAF genes.
Conclusion: The initial results of our research suggest that NRAS gene mutations — particularly at codon 13 — are
increasingly recognized as contributors to resistance in the context of rechallenge therapy. When examining tissue
samples taken before the previous line of anti-EGFR therapy, the prevalence of NRAS mutations is notably low,
ranging from 3% to 5% (7). Although these findings need validation in larger patient cohorts, they indicate that
NRAS mutations could be a key factor in the development of resistance to the anti-EGFR treatments. The occurrence
of multiple mutations in individual patients underscores the genetic heterogeneity of CRC, which represents a key
hallmark of the disease and may play a pivotal role in the development of therapeutic resistance. The underlying
mechanisms may involve alterations in the mutational landscape that promote the emergence of secondary resistance
mutations, as well as selective pressure induced by anti-EGFR therapies, which allow previously minor clones to persist
(8). Additionally, we observed that 72% of patients still harbor at least one mutation in the KRAS or NRAS gene even
after 4 months without therapy. The higher percentage of mutations acquired after receiving cetuximab compared
to panitumumab also provides an opportunity to further investigate the role of KRAS/NRAS/BRAF mutations in the
acquisition of resistance to both drugs in patients with mCRC. Ultimately, rechallenging with the same anti-EGFR
antibody-mAb-based therapy at the right time may offer new clinical benefits for mCRC patients, potentially delaying
disease progression and improving treatment outcomes.

Acknowledgments and funding: We would like to express our gratitude to Merck, who provided RAS tests for the
benefit of patients and enabled molecular testing of mCRC patients who were candidates for EGFRi rechallenge. The
results of these tests have been utilized in our research.

References:

1. Mariani S, Puzzoni M, Giampieri R, Ziranu P, Pusceddu V, Donisi C, et al. Liquid Biopsy-Driven Cetuximab
Rechallenge Strategy in Molecularly Selected Metastatic Colorectal Cancer Patients. [Internet]. Front Oncol. 2022
Apr 21 [cited 2025 Aug 30];12. Available from: https://www.frontiersin.org/journals/oncology/articles/10.3389/
fonc.2022.852583/full
2. Mendelsohn J, Prewett M, Rockwell P, Goldstein NI. CCR 20th Anniversary Commentary: A Chimeric Antibody,
C225, Inhibits EGFR Activation and Tumor Growth. Clin Cancer Res. 14. Jan 2015.;21(2):227–9.
3. Santini D, Vincenzi B, Addeo R, Garufi C, Masi G, Scartozzi M, et al. Cetuximab rechallenge in metastatic colorectal
cancer patients: how to come away from acquired resistance? Ann Oncol 2012 Sep;23(9):2313–8.

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