Resistance mechanisms to anti-EGFR therapy in RAS/RAF wildtype colorectal cancer varies by regimen and line of therapy.
Authors: Parseghian Christine Megerdichian , Sun Ryan , Woods Melanie Nicole , Napolitano Stefania , Alshenaifi Jumanah , Willis Jason , Nunez ShaKayla Kentel , Sorokin Alexey , Kanikarla Marie Preeti , Raghav Kanwal Pratap Singh , Morris Van K. , Shen John Paul Y.C. , Vilar Sanchez Eduardo , Rehn Marko , Ang Agnes , Troiani Teresa , Kopetz Scott 
Published: June 2022

3554 Background: The conventional theory for the development of treatment resistance to anti-EGFR for metastatic colorectal cancer (mCRC) is the selective growth advantage of pre-existing therapy-resistant subclones with genomic mechanisms such as RAS mutations, leading to treatment resistance and disease progression. However, the impact of cytotoxic chemotherapy in combination with anti-EGFR on the mechanisms of resistance has not been assessed. Methods: We analyzed paired plasma samples from RAS/BRAF/EGFR wild-type mCRC patients enrolled in three large randomized phase 3 trials of anti-EGFR rechallenge in whom paired baseline and time of progression plasma samples had been collected for sequencing of ctDNA on a platform optimized for very low allele frequencies. 569 patients had paired baseline and progression ctDNA samples analyzed, including 147 in the first line study of FOLFOX +/- panitumumab, 91 patients in third line with panitumumab vs best supportive care, and 331 patients in the third line study of cetuximab vs. panitumumab. The mutational signature of the alterations acquired with therapy was evaluated. We also established colon cancer cell lines with resistance to cetuximab, FOLFOX, and SN38, and profiled transcriptional changes. Results: Using serial plasma samples, we demonstrate that patients whose tumors were treated with and responded to anti-EGFR alone were approximately 5-times more likely to develop acquired mutations at progression compared to those treated with an EGFR inhibitor in combination with cytotoxic chemotherapy (46% vs. 9%, respectively; p < 0.001). Consistent with this clinical finding, cell lines with non-genomic acquired resistance to cetuximab were cross-resistant to cytotoxic chemotherapy and vice-versa, with transcriptomic profiles consistent with epithelial to mesenchymal transition. In contrast, common acquired genomic alterations in the MAPK pathway that drive resistance to EGFR monoclonal antibodies do not impact sensitivity to cytotoxic chemotherapy. Further, contrary to the generally accepted hypothesis of clonal expansion of acquired resistance, in our work we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at the time of progression (8%), and most remained subclonal (44%) or disappeared (49%). Conclusions: Collectively, this work outlines a model of resistance where non-genomic mechanisms of resistance common to both EGFR inhibitors and cytotoxic chemotherapy predominate in patients treated with EGFR and chemotherapy combinations. With EGFR inhibitor monotherapy, genomic acquired resistance mechanisms predominate, although only rarely through expansion of pre-existing subclones. These findings have important implications for strategies of EGFR-inhibitor rechallenge studies.