Page 138 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

analysis. LC-MS/MS profiling of PE-derived vesicles using the Norgen kit with additional pretreatment with Proteinase
K (as in ExoProK) or a CK-SEC combination to enhance purity may help establish PE-derived EVs as a valuable source for
biomarker discovery in LC.

Acknowledgments and funding: This study was funded by the Horizon Europe EXPAND_EV (HORIZON-MSCA-2023-
SE-01, Grant Agreement No. 101182851) and the Ministry of Science, Technological Development and Innovation of
the Republic of Serbia (Agreement No. 451-03-136/2025-03/ 200043).

P45

Liquid Biopsy as a Tool in Guiding Anti-EGFR Monoclonal Antibody Rechallenge Therapy in Metastatic
Colorectal Cancer

Vanja Stevanović1, Ana Damjanović1, Neda Nikolić1, Nevena Tišma-Miletić1, Milena Čavić1, Marija Ristić1, Miroslav
Kreačić2, Sanja Kostić3, Nebojša Manojlović4, Marina Marković5, Biljana Kukić6, Miljana Džunić7, Radmila Janković1

1Institute for Oncology and Radiology of Serbia; Belgrade, Serbia
2Clinical Hospital Center Zemun; Belgrade, Serbia
3Clinical Hospital Center Bezanijska kosa; Belgrade, Serbia
4Military Medical Academy; Belgrade, Serbia
5Department of Oncology, University Clinical Center Kragujevac; Kragujevac, Serbia
6Oncology Institute of Vojvodina; Sremska Kamenica, Serbia
7University Clinical Center Nis; Nis, Serbia

Keywords: metastatic colorectal cancer (mCRC), liquid biopsy (LB), anti-EGFR antibodies, rechallenge therapy

Background: In patients with metastatic colorectal cancer (mCRC) who progressed to anti-EGFR monoclonal antibody
(mAb) therapy, such as cetuximab and panitumumab, a rechallenge with the same agents is becoming a promising
approach. Although patients demonstrate objective progression during the anti-EGFR treatment, emerging findings
suggest that anti-EGFR-resistant clones decay, enabling the possibility of a rechallenge strategy (1). A key step for a
deeper understanding of clinical progression in patients is the identification of mechanisms of secondary resistance
to anti-EGFR mAb therapies. Limited data are currently available on the resistance mechanism that develops during
the anti-EGFR treatment. Intratumoral heterogeneity is assumed to play a significant role in mCRC progression. During
anti-EGFR therapy, subclones continuously arise through genomic mutations, resulting in faster disease progression
and poorer overall survival (OS) in patients (2). Santini et al. (3) demonstrated that the reuse of cetuximab was a
potentially effective strategy in patients with mCRC who had previously achieved clinical benefit from cetuximab-based
therapy. A sufficiently lengthy treatment-free period, of a minimum of four and optimally longer than six months,
should be provided. This study was the first to lay the groundwork for the hypothesis that acquired resistance to anti-
EGFR therapy may be reversible and that, after a break from the therapy, the tumor may become sensitive again, which
improves subsequent response and increases patient free survival (PFS). European and American health authorities
have restricted the use of cetuximab and panitumumab, as mono- or in combination with chemotherapy, only to
patients with wild-type (wt) RAS gene status (4). As with first-line treatment, confirmation of wt KRAS/NRAS gene
status is recommended for anti-EGFR mAb reintroduction (5). Mutations in the BRAF gene are also recognized as
negative prognostic markers in the response to the anti-EGFR therapy, and according to the European Society For
Medical Oncology (ESMO) guidelines, testing for mutations in the BRAF gene is also recommended (6). The preferred
method for selecting patients for the rechallenge strategy, but also for detecting mutations and monitoring secondary
resistance, is liquid biopsy (LB). LB is a minimally invasive technique that shows promise in identifying various molecular
changes crucial for achieving an effective response to treatment.
In our research, we aimed to analyze the distribution of mutations and characteristics of circulating tumor DNA (ctDNA)
in plasma samples from patients who developed resistance to the anti-EGFR antibodies. This study may enhance our
understanding of the mechanisms underlying the evolution of resistance to targeted therapies.
Materials and methods: Samples were collected from 25 patients (17 male/8 female), potential candidates for
rechallenge therapy, over a period of 7 months. The median age of the study population was 64 years (range: 40-
80 years). Among the 25 patients enrolled, 15 received cetuximab and 10 received panitumumab, before disease
progression. Patients were included if the previous anti-EGFR therapy had been applied for at least 6 months. According
to Santini's hypothesis (3), blood was sampled at least 4 months after receiving the last mAb-based therapy. The blood

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