PURPOSE Acquired resistance to anti-EGFR therapy (EGFRi) in CRC has previously been explained by the model of acquiring new mutations in KRAS/NRAS/EGFR, among other MAPK-pathway members. However, this was primarily based on single-agent EGFRi trials and little is known about the resistance mechanisms of EGFRi combined with effective cytotoxic chemotherapy in previously untreated patients. METHODS We analyzed paired plasma samples from RAS/BRAF/EGFR wild-type mCRC patients enrolled in three large randomized trials evaluating EGFRi in the first-line in combination with chemotherapy and as a single-agent in third-line. The mutational signature of the alterations acquired with therapy was evaluated. CRC cell lines with resistance to cetuximab, FOLFOX, and SN38 were developed, and transcriptional changes profiled. RESULTS Patients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared to those treated in combination with cytotoxic chemotherapy (9%). Further, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition (EMT). In contrast, commonly acquired resistance alterations in the MAPK pathway do not impact sensitivity to cytotoxic chemotherapy. CONCLUSION These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

3520 Background: In RAS/RAF WT colorectal cancer (CRC), rechallenge with anti-EGFR therapy (EGFRi) in patients (pts) with prior response leads to clinical benefit, with response rates up to 30% in prior trials. However, secondary MTs in the MAPK signaling pathway have been implicated in resistance to EGFRi. We designed a phase 2 trial to evaluate the efficacy of EGFRi rechallenge +/- a MEK inhibitor (trametinib) based on pre-treatment ctDNA MTs. Methods: This trial evaluated the efficacy and safety of EGFRi rechallenge +/- trametinib in pts with RAS/BRAF WT, MSS, treatment refractory mCRC who achieved clinical benefit with prior EGFRi based therapy for ≥16 weeks with subsequent progression. Pre study ctDNA was used to enroll in one of 3 arms: Arm A: Pts with an acquired EGFR ECD MT but absence of RAS/BRAF/MAP2K1 or with absence of any acquired resistance MT (Arm C) at time of study initiation received panitumumab 6 mg/kg IV Q2 wks. Arm B: Pts with an acquired RAS/BRAF/MAP2K1 MT received panitumumab 4.8 mg/kg plus trametinib 1.5 mg PO daily. Pts in Arms A and C were allowed to cross over on progression. The primary endpoint was ORR by RECIST v1.1. Results: 54 pts were enrolled, with 52 evaluable for efficacy. Median age is 59 yrs (range, 37-78), and 23 (46%) are female. Median number of prior therapies was 3. Three, 20, and 31 pts were enrolled in Arms A, B, C, respectively. Grade 3 TREAs occurred in 29 (54%) pts (all receiving the doublet regimen) and included acneiform rash in 17 (31%) and others occurring in < 5% of pts. There were no grade 4 TRAEs. In pts with no acquired MTs (Arm C), ORR was 20% (6/30) (95% CI, 0.07-0.37), DCR 67% (20/30) (95% CI, 0.45- 0.81), and median PFS and OS 4.1 mo and 11.2 mo, respectively. The median DOR was 5.5 mo. 22 patients crossed over to add trametinib at time of progression, without any responses. In contrast, in pts with acquired RAS/RAF/MAP2K1 MTs (Arm B), there were no responses, with DCR of 63% (12/19) (95% CI, 0.36-0.81), and median PFS and OS 2.1 mo and 5.9 mo, respectively. Only 3 pts were identified with EGFR ECD MTs (Arm A), and ORR is 0% (0/3) in this cohort, with DCR 67% (2/3) (95% CI, 0.09-0.99). Pts with PR had a longer median interval from prior EGFRi and longer time on prior EGFRi than those with SD+PD (5.5 vs 3.6 mo; p = 0.03, and 9.5 vs. 8.8 mo; p = 0.03, respectively). Conclusions: CtDNA guided rechallenge leads to responses in 20% of pts without acquired resistance MTs, with DCR of 67%. This exceeds current third line standard options. While panitumumab has the potential to block EGFR ECD mutations arising from cetuximab, these mutations in isolation were uncommon and there were no signals of efficacy. Although the acneiform rash induced by the combination of MEK and EGFR inhibition was manageable with close dermatologic management, the combination failed to improve outcomes for pts with acquired resistance. Alternative approaches to downstream MAPK blockade should be explored to improve outcomes. Clinical trial information: NCT03087071.

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607 Background: Microsatellite instability (MSI-H) and increased tumor mutation burden (TMB) have been associated with clinical benefit from immunotherapy. A small subset of patients with intrahepatic cholangiocarcinoma (iCCA) have microsatellite instability (MSI-H). Understanding their clinical outcomes and genomic landscape has been limited. These patients may derive clinical benefit from immunotherapy compared to chemotherapy. Methods: 7565 cases of iCCA underwent comprehensive genomic profiling of 324 genes (FoundationOne CDx). Prevalence of genomic alterations in cases with MSI-H were compared with MSI-low and MSS iCCA using Fischer’s exact test with Bonferonni correction. Clinical and pathological data were collected by retrospective chart review for patients with MSI-H/dMMR iCCA. Results: Among 7565 iCCA cases identified, 137 (1.8%) were MSI-H. Compared to MSS/MSI-low cases, MSS-H patients were more likely to be male and have Lynch syndrome. Median TMB was 21.7 (muts/Mb). MSI-H cases were enriched for genomic alterations (GA) in APC (13.9%), TP53 (59.9%), ARID1A (13.9%), and PRBM1 (37.2%). MSI-H cases had lower frequencies of previously identified, actionable iCCA drivers such as FGFR2 fusions (0% v 9%, p=NS), IDH1 (3.7% v 14.5%, p<0.0001) and IDH2 (0% v 4.1%, p=0.007). Compared to MSS/MSI-low, MSI-H cases had higher GA in PIK3CA (16.8% v 6.6%, p=<0.0001), PTEN (10.9% v 2.8%, p=<0.0001), and BRCA2 (10.2% v 2%, p=<0.0001). MSI-H cases had lower GA in CDKN2A (24.1% v 30.9%, p=0.09) and MTAP loss (3.4% v. 15.5%, p<0.0001). Five patients were identified with dMMR/MSI-H iCCA out of 974 patients at a single institution (0.5%). Median age at diagnosis was 53 years (range 46-70) and 83% were male (n=4). Two patients had resectable disease at time of diagnosis. TMB range was 20-29 (muts/Mb). Patients had 4-6 co-mutations on average with the most common GA being TP53, ARID1A and CDKN2A. One patient had Lynch syndrome with germline alteration of MLH1 gene and one patient had a germline BRCA mutation. One patient was noted to have FGFR2 rearrangement. Gemcitabine/Cisplatin was given to 3 of 6 patients as first-line (1L) therapy, with median progression free survival of 4 months. One hundred percent of patients received immunotherapy (IO) as first or second-line (2L) therapies. Patients receiving IO in the 1L had responses ranging from 4 to 9 months. Three patients have ongoing responses. Median overall survival ranged from 12 months to >18 months. Two patients presented with CNS involvement and did not respond to immunotherapy-based regimens. Conclusions: MSI-H represents a small subset of patients with iCCA. High-TMB noted amongst MSI-H iCCA. Currently, MSI testing is not universally performed and may be necessary to identify and best treat these patients. These findings support the development of personalized immunotherapy treatment strategies for this distinct patient population.

4090 Background: BTC is associated with poor prognosis and limited treatment choices. There is limited evidence on HCRU and costs among BTC patients receiving systemic treatments (ST). This study examined HCRU and total direct cost of care among BTC patients treated with ST to understand the disease burden in management of BTC. Methods: A retrospective cohort study of BTC patients who received first line (1L) ST was conducted, using US private payer Cancer Care Quality Program data and administrative claims from the HealthCore Integrated Research Database between 07/01/2014 - 03/31/2021. Patients with ampullary cancer, brain/ CNS metastases, other primary cancer before BTC diagnosis were excluded. Per patient per month (PPPM) costs in 2020 USD were calculated during 1L, 2L, and 3L treatments associated with HCRU from inpatient, emergency room, and outpatient visits as well as outpatient pharmacy dispensing. Results: Among 298 BTC patients (biliary tract, n=203; gallbladder, n=65; bile duct, n=30; stage IV, n=231; stage III, n=28; stage I/II, n=39) who received ST, mean (SD) age was 61.7 (9) years at 1L treatment initiation, and the majority were female (58%). Following 1L treatment, 44% received 2L treatment, and 16% received 3L treatment. Median follow-up was 7.6 months. Among 201 (67%) patients who had hospitalization in the follow-up period, mean (SD) number of hospitalizations was 2.5 (2), and the average length of stay was 7.0 (5) days. Total PPPM all-cause costs were the lowest during 1L treatment (mean [SD]: $19,589 [$22,603]), and increased as the treatment advanced (2L: $22,617[$19,302]; 3L: $33,534[$40,588]). Similar trend was observed in BTC-related total costs with $16,237 ($22,452) in 1L, $19,083 ($18,670) in 2L and $27,609 ($39,949) in 3L. The table summarizes BTC-related HCRU and PPPM cost during each line of therapy. Conclusions: Study findings suggest significant resource use burden and high total direct medical costs for BTC patients receiving ST. Hospitalizations and outpatient visits represent important HCRU and cost for BTC. These data indicate a need for future newer innovative therapies in the management of BTC.[Table: see text]