Search

129 Background: Rechallenge therapy with epidermal growth factor receptor (EGFR) inhibitors in chemo-refractory metastatic colorectal cancer (mCRC) is an emerging therapeutic approach. Trifluridine-tipiracil is approved for treatment of chemo-refractory mCRC patients. Methods: Chemo-refractory RAS WT mCRC patients, that had a major response (partial or complete response) to first-line chemotherapy plus an anti-EGFR monoclonal antibody and had an-anti-EGFR drug-free interval during second-line therapy of four or more months were randomized in a phase II trial to assess the addition of the anti-EGFR monoclonal antibody panitumumab to trifluridine-tipiracil as third-line rechallenge therapy. The primary endpoint was progression free survival (PFS). Baseline plasma was analyzed for circulating free tumor (ct) DNA by using Biocartis Idylla platform to detect mutations in KRAS, NRAS, BRAF ( V600E) and EGFRextracellular domain ( S492R). In 24 patients with baseline RAS/BRAF wild type (WT) ctDNA, Foundation One liquid CDx (324 gene profiling) was also performed before treatment and at disease progression. Results: 62 patients were treated with trifluridine-tipiracil (31 patients, arm A) or with trifluridine-tipiracil plus panitumumab (31 patients, arm B). As of September 16, 2022, 1 patient in arm A and 2 patients in arm B were on treatment. The primary endpoint was met. Median PFS (mPFS) was 4 months in arm B versus 2.5 months in arm A [hazard ratio (HR): 0.48; 95% CI 0.28-0.82; P = 0.007]. Baseline plasma RAS/BRAF WT ctDNA was found in 23/31 patients in Arm A and in 26/31 patients in Arm B. In this group, mPFS was 4.5 months in Arm B versus 2.6 months in Arm A (HR: 0.48; 95% CI 0.26-0.89; P = 0.019). Disease control (major responses plus stable disease) was higher for patients in Arm B compared to Arm A (81% versus 48%), whereas disease progression was the best response in 19% versus 52% patients, respectively. PFS rates at 6 and 12 months were 38.5% and 15.4% in arm B versus 13% and 0% in arm A. At disease progression, Foundation One liquid CDx detected several mutations within the EGFR pathway, which could correlate with cancer cell resistance to panitumumab. Conclusions: This is the first prospective randomized trial which evaluated anti-EGFR monoclonal antibody (panitumumab) in addition to standard of care (trifluridine-tipiracil) as third-line rechallenge therapy in chemo-refractory RASWT mCRC patients. Baseline liquid biopsy allows selection of RAS/BRAF WT ctDNA patients who could have a relevant clinical benefit. Clinical trial information: NCT05468892 .

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.

145 Background: Based on promising results from latest trials, a crucial point is to evaluate whether encorafenib (E) plus cetuximab (C), alone or in combination with chemotherapy, can improve clinical outcomes relative to current standard of care in previously untreated BRAFV600E mutant mCRC. Considering the high number of BRAFV600E mutant mCRC patients who will never receive a second-line treatment, the rationale of this strategy is to maximize treatment outcome within the first-line setting. Methods: We performed an in vivo study using human BRAFV600E CRC cell line-derived xenografts in nude mice. We evaluated the efficacy of encorafenib (E) + cetuximab (C), FOLFOX, and FOLFIRI, both as individual regimens and in combinations. Mice were treated for 3 weeks and followed for an additional 8 weeks to evaluate durability of tumor control. Additionally, we validated our findings using 3 BRAFV600E mutated patient derived xenografts. Tumors progressing on single agent and combined treatment were profiled by RNA sequencing, protein extraction for RPPA/Western blot, and establishment of in vitro primary cell cultures for further analyses. Results: Our study showed across all 4 models both FOLFOX and FOLFIRI, each in combination with encorafenib plus cetuximab, having greater efficacy than encorafenib plus cetuximab or either chemotherapy alone. No significant change in toxicity was seen with the addition of chemotherapy. Interestingly, in the one model with long term treatment, FOLFOX + E +/- C performed greatest over the long-term, with significant endpoint separation against all other treatment arms (P < 0.05). Conclusions: Taken together, results from our study suggest that the addition of chemotherapy to BRAF+EGFR targeted therapy can further increase the magnitude of response in BRAFV600E mCRC and is a promising combination now being explored clinically. Additionally, this research will substantially contribute to our understanding of the genetic and molecular bases of resistance to target therapies and chemo-based approach in BRAFV600Econtext.

3578 Background: Promising antitumor activity of so called rechallenge treatment with anti-epidermal growth factor receptor (EGFR) drugs in patients with RAS wild type (RAS WT) metastatic colorectal cancer (mCRC) has been recently reported. Beside the absence of resistance mutations at plasma circulating tumor DNA (ctDNA) analysis, no biomarkers of response to anti-EGFR rechallenge strategy have been identified. Methods: We conducted the single arm phase II CAVE mCRC trial to evaluate the combination of cetuximab as rechallenge plus avelumab treatment in 77 RAS WT mCRC patients, with complete (CR) or partial response (PR) to first line chemotherapy plus anti-EGFR drugs, who developed acquired resistance and received a subsequent line of therapy. A post-hoc baseline analysis of circulating tumor DNA (ctDNA) for KRAS, NRAS, BRAF and EGFR-S492R mutations was performed for 67/77 (87%) patients. The correlation of skin toxicity (ST) and other clinical variables with OS, PFS and response rate (RR) was assessed. Results: Cetuximab plus avelumab provided in the intention to treat population (ITT) median overall survival (mOS) of 11.6 months [95% Confidence Interval (CI), 8.4-14.8] and median PFS (mPFS) of 3.6 months (95% CI, 3.2-4.1) with a manageable toxicity profile. Thirty-three (42.9%) patients experienced grade 2-3 ST with mOS of 17.8 months (CI 95% 14.9-20.7), whereas 44 (57.1%) patients with grade 0-1 ST exhibited mOS of 8.2 months (CI 95% 5.6-11), (HR 0.51, CI 95% 0.29-0.89, P = 0.019). mPFS was 4.6 months (CI 95% 3.5-5.8) in patients with grade 2-3 ST, compared to 3.4 months (CI 95% 2.8-4.1) in patients with grade 0-1 ST (HR 0.49, CI 95% 0.3-0.8, P = 0.004). Grade 2-3 ST and baseline RAS/BRAF/EGFR WT ctDNA were the only variables with statistically significant effect on OS both at univariate and multivariate analyses. ST, number of metastatic sites ≤2, surgery of primary tumor and RAS/BRAF/EGFR WT ctDNA were associated with an improvement in PFS only at univariate analysis. In the 33 patients with grade 2-3 ST, 1 (3%) CR, 2 (6.1%) PR and 24 (72.7%) stable disease (SD) were observed, with disease control rate (DCR) of 81.8%. In the 44 patients with grade 0-1 ST 0 CR, 3 (6.8%) PR, 20 (45.5%) SD, with 52.3% DCR were reported. Conclusions: Cetuximab plus avelumab is effective and well tolerated as rechallenge treatment in mCRC. ST is a clinical biomarker for the identification of RAS/BRAF mCRC patients that could benefit from anti-EGFR rechallenge. Clinical trial information: NCT04561336.

3594 Background: Recently, in metastatic colorectal cancer (mCRC), the detection of RAS mutations by circulating tumor (ct) DNA has recently emerged as a valid and non-invasive alternative approach, overall showing a high concordance with the standard tissue genotyping, giving information on response to EGFRi treatment and resistant mechanisms. However, RAS mutations may be missed due to low levels of any ctDNA in the blood (false-negative), and it has been difficult to distinguish this from patients without a RAS mutation in the tumor (true-negative). We propose a methodology that can be applied to multi-gene ctDNA testing panels to accurately distinguish true- and false-negative tests. Methods: 357 subjects with tissue and multi-panel ctDNA testing from MD Anderson (MDACC) were used as a training dataset and 295 subjects from Massachusetts General Hospital (MGH) dataset as the testing dataset. CtDNA panels contained between 65 and 70 genes, allowing evaluation of tumor ctDNA shedding from variant allele fraction (VAF) levels in the plasma from other genes (such as APC and TP53). Based on the relationship between KRAS and the VAFs of other gene, we established a Bayesian model providing a posterior probability of false negative in the ctDNA test, using thresholds of < 5% (low), 5-15% (medium), and > 15% (high). This model was validated on the MGH database. Results: Across both cohorts, 431 patients were ctDNA wild type for KRAS. Of those, 29 had tissue documenting a KRAS mutation for a false negative rate of 8%. The model provides the posterior probability that a KRAS mutation is indeed present in the tissue given the observed values of allele frequencies for other mutated genes in the plasma. In the validation cohort, a predicted low false negative had no false negatives (0/62, 95% CI 0%-5.8%), while a predicted medium false negative rate was associated with 3% false negative (1/32, 95% CI 0%-16%). In contrast, a high predicted false negative rate was associated with 5% false negative (5/100, 95% CI 1.6%-11%). The results demonstrate the ability of our tool to discriminate between subjects with true negative and false negatives, as a higher proportion of false negatives are observed at higher posterior probabilities. Conclusions: In conclusion, our approach provides increased confidence in KRAS ctDNA mutation testing in clinical practice, thereby facilitating the identification patients who will benefit from EGFR inhibition while reducing the risk of false negative tests. Extension of this methodology to NRAS and BRAF is possible, with clinical application enabled by a freely available online tool.

3514 Background: Colorectal cancers (CRC) lacking RAS MTs treated with EGFRi are thought to evolve by a repetitive process of genetic diversification and clonal evolution. Acquired MTs in KRAS, NRAS, BRAF, MAP2K1, and EGFR are known mechanisms of acquired resistance in the EGFRi refractory population. However, the prevalence of MTs in the first line (1L) setting is not well established as most experience with EGFRi has been beyond the 1L setting. Methods: We analyzed paired plasma samples from RAS/BRAF/EGFRWT mCRC patients (pts) enrolled in 3 large randomized phase 3 trials who had been treated with EGFRi and in whom paired baseline (BL) and time of progression (PRO) plasma samples had been collected for sequencing of ctDNA on a platform optimized for very low allele frequencies (Plasma Select-R™ and Resolution Bio™). Prevalence of MTs at BL and PRO from a 1L study (‘203; FOLFOX ± panitumumab) were compared with 2 studies in the third line setting (3L; ‘007; panitumumab + best supportive care [BSC] vs BSC; and 3L; ‘763; panitumumab vs. cetuximab), to assess the frequency of acquired resistance mutations via ctDNA analysis. Results: For pts with available paired plasma samples (n = 112 for ‘203; n = 89 for ‘007; n = 274 for ‘763), acquisition of at least one KRAS, NRAS, BRAF, MAP2K1, or EGFR MT was significantly less common in post-progression samples in the EGFR containing arms of the 1L ‘203 study compared to the 3L ‘763 and ‘007 studies (6.8% vs 50.4% vs 39.6%, respectively; p < 0.001). In the non EGFR containing arms of the ‘203 and ‘007 study, the rate of acquired MTs was 7.5% and 0%, respectively (p = 1). While this difference in the rate of acquired MTs between the EGFR and non EGFR containing arms was statistically significant for the 3L study (p < 0.001) it was not significant for the 1L study. Further, pts on both 3L studies treated with EGFRi who experienced CR, PR or SD acquired more MTs than those who had PD as best response (53.6% vs 33.3%, respectively; p < 0.001). This relationship was not significant in the 1L setting (7.7% vs 0%; p = 1). Subclonal MTs (rMAF < 25%) in KRAS, NRAS, EGFR, BRAF and MAP2K1 were present at BL in 129 pts (27%). Based on the hypothesis that EGFRi is selecting for rare existing mutated cells in the tumor, we would expect expansion of any preexisting subclones in the BL samples. However, in contrast to expectations, these subclones rarely expanded to become clonal at the time of progression (12.4%). Conclusions: In contrast to expectations, acquired KRAS, NRAS, BRAF, EGFR, or MAP2K1 MTs rarely develop after 1L therapy. While selective pressure appears to increase the frequency of acquired MTs in the 3L setting, preexisting subclonal MTs do not appear to be the dominant source of acquired MTs at progression, implying that there may also be a transient mutational process driving resistance rather than expansion of preexisting clones. These findings have significant implications for ongoing and planned EGFRi rechallenge studies.

110 Background: BRAFV600E mutation identifies mCRC patients with poor prognosis with only little benefit from standard therapy. Analysis from TCGA revealed that 89% of BRAFV600E CRC tumors were associated with a high CpG island methylator phenotype (CIMP-H), which may result in epigenetic silencing of tumor suppressor genes, while only 29% of BRAF wild-type tumors are CIMP-H. In this study, we define key pathways regulated by global DNA hypermethylation in the context of BRAFV600E mutation. Methods: We analyzed the TCGA Illumina 450k array methylation datasets and RNA-sequencing datasets for 97 CIMP-H CRC tumors (27 BRAFV600E; 69 BRAF WT) identified by five universal CIMP annotations ( p14, p16, MLH1, MINT1, MINT2, MINT31). We defined differential methylation profile according to BRAF mutation status and calculated Spearman correlation between methylation and gene expression to identify CIMP-H BRAF-associated genes. Next, pathways enriched with CIMP-H BRAFV600E tumors were defined using PANTHER pathway analysis. Additionally, β-catenin IHC were conducted on 145 MD Anderson CRC patient samples and compared by CIMP and BRAF status. Results: BRAF mutation is associated with lower rates of APC mutation as has previously been shown (32%, 82%). We identified 6,097 differentially methylated probes by BRAF mutation status (FDR = 10-4), and as expected, our data suggests a higher methylation profile in BRAFV600E mutated tumors compared to BRAF WT. Intriguingly, CIMP-H BRAF-associated genes showed enrichment in the Wnt-signaling and cadherin signaling pathways ( p< 0.0001 (FDR < 0.0001)). Despite the epigenetic Wnt-signaling, nuclear β-catenin expression (as a measure of Wnt activity) in CIMP-H and BRAF tumors remains lower than for non-CIMP, and BRAF wild-type ( p= 0.0003 for comparison of CIMP). Conclusions: Genes under methylation regulation in the BRAF-mutant context showed enrichment in Wnt-signaling pathway. Since BRAFV600E CRC tumors have a low association with APC mutation, this data suggests role of epigenetic regulation of the Wnt-pathway activation. However, as measured by nuclear β-catenin, Wnt activation in these tumors is not as high as traditional APC-mutated CRC tumors. CIMP-H tumors with BRAFV600E mutation is a unique subset of CRC tumor that have Wnt-pathway activation regulated by epigenetic modifications more than a β-catenin activation.