RAS Wt Research Articles

11P - Triple blockade of EGFR, MEK and PD-L1 as effective antitumor treatment in PD-L1 overexpressing, MEK inhibitor resistant colon cancer cells

BackgroundMolecular mechanisms driving acquired resistance to anti-EGFR therapies in metastatic Colorectal Cancer (mCRC) are complex but generally involve the activation of the downstream RAS-RAF-MEK-MAPK pathway. MethodsIn order to understand the mechanism underlying MEK inhibitor (MEKi) resistance, we generated three different MEKi resistant models, two all RAS WT (SW48-MR and LIM1215-MR) and one mutated in RAS (HCT116-MR). ResultsThese models showed features related to the gene signature of Colorectal Cancer CMS4 with upregulation of immune pathway as confirmed by Microarray and Western blot analysis. In particular, moving forward the MEKi phenotype we assisted to the loss of epithelial features and acquisition of mesenchymal markers and morphology. Moreover, this change in the morphology is accompanied by up-regulation of PD-L1 expression and activation of EGFR and its downstream pathway, independently of cell line status mutation. To extend these in vitro findings, we performed an in vivo study using MC38 and CT26 MEKi resistant syngeneic models that we have previously generated. Combined treatment of MEKi, EGFR inhibitor (EGFRi) and PD-L1inhibitor (PD-L1i) resulted in a marked inhibition of tumor growth in both MC38–MR and CT26-MR xenograft model. ConclusionsThese results suggest a strategy to potentially overcome immunotherapy resistance in CMS4-like tumors and to improve the efficacy of MEK inhibition by co-treatment with other agents providing an additional therapeutic strategy via modulation of host immune responses. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

533PD - BACCI: A phase II randomized, double-blind, multicenter, placebo-controlled study of capecitabine (C) bevacizumab (B) plus atezolizumab (A) or placebo (P) in refractory metastatic colorectal cancer (mCRC): An ACCRU network study

BackgroundThe benefit of immune checkpoint blockade in mCRC is currently limited to mismatch repair (MMR) deficient tumours. Increasing evidence suggests that the vascular endothelial growth factor (VEGF) pathway plays a role in cancer immune evasion. Co-targeting the PD-1/PD-L1 and VEGF axes may result in clinical activity in mCRC. Methods133 patients (pts) were randomized 2:1 to receive C/B/P (Arm A) or C/B/A (Arm B), stratifying by ECOG and RAS. Pts had progressed on 5-FU, oxaliplatin, irinotecan, bevacizumab, and anti-EGFR therapy (if RAS wt). Prior anti-PD-1/PD-L1 therapy was not permitted. Doses were C 850 or 1000mg/m² d1-14, B 7.5mg/kg d1, and A 1200mg d1 in 21day cycles. Primary endpoint was progression free survival (PFS). 110 events were required to detect PFS hazard ratio (HR) of 0.65 at 1-sided α=0.1 with 80% power. Secondary endpoints were objective response rate (ORR), overall survival (OS), and safety/tolerability. Primary and secondary efficacy analyses were performed using modified intent to treat analysis (mITT). ResultsAnalysis includes 46 pts (Arm A) and 82 pts (Arm B) with median age 58 yo, 40% female, 47% ECOG 0. As of 4/12/19, median follow-up was 12.35 months (mo). Proficient MMR or microsatellite stable (MSS) is present in 86.7% vs 85.7%. In mITT analysis, median PFS is 3.3mo (2.1-6.2) vs 4.4mo (4.1-6.4) with a HR of 0.725 (0.491-1.07), 1-sided log-rank p-value 0.051. In MSS only pts, HR for PFS is 0.67 (0.44-1.03). ORR is 4.35% (0.5-14.8) vs 8.54% (3.5-16.8), p=0.5. The 12mo OS is 43% (29-63) vs 52% (42-65), HR 0.94 (0.56-1.56), p=0.4. Most common grade>= 3 related adverse events are hypertension (7% vs 9%), hand-foot syndrome (4% vs 6%), and diarrhea (2% vs 7%). ConclusionsThe study reached its prespecified primary endpoint and the addition of A to CB results in a significantly longer PFS. No new or increased safety signals are identified. Further investigation in a larger phase III study may be warranted. This is the first positive study co-targeting PD-1/PD-L1 and VEGF axes in mCRC and correlative analyses may help identify predictors of benefit. Clinical trial identificationNCT0287319. Legal entity responsible for the studyACCRU. FundingGenentech Roche. DisclosureH.J. Lenz: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Genentech Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb. P.M. Boland: Research grant / Funding (institution): Merck; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Hemispherx; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Isofol Medical; Research grant / Funding (institution), Travel / Accommodation / Expenses: Ipsen; Research grant / Funding (institution): Athenex; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Clinical Genomics. A.B. Nixon: Research grant / Funding (self): Seattle Genetics; Research grant / Funding (self): MedPacto; Research grant / Funding (self): Genentech Roche; Advisory / Consultancy, Research grant / Funding (self): Tracon Pharma; Research grant / Funding (self): Acceleron Pharma; Research grant / Funding (self): Leadiant Biosciences; Research grant / Funding (self): Sanofi-Aventis; Advisory / Consultancy: Eli Lilly. H. Hurwitz: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech Roche. M.G. Fakih: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Amgen; Advisory / Consultancy: Array; Advisory / Consultancy: Bayer; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Novartis. All other authors have declared no conflicts of interest.

584P - Noninferiority on overall survival of every-2-weeks vs weekly schedule of cetuximab for first-line treatment of RAS wild-type metastatic colorectal cancer

584P - Noninferiority on overall survival of every-2-weeks vs weekly schedule of cetuximab for first-line treatment of RAS wild-type metastatic colorectal cancer

LBA90 - Primary analysis results of randomized controlled trial evaluating reactive topical corticosteroid strategies for the facial acneiform rash by EGFR inhibitors (EGFRIs) in patients (pts) with RAS wild-type (wt) metastatic colorectal cancer (mCRC): FAEISS study

BackgroundThe management strategies regarding the strength of reactive topical corticosteroids have not been well evaluated in clinical trials. This FAEISS study is designed to confirm the superior efficacy of reactive topical corticosteroid strategies with serially ranking-DOWN from very strong levels compared with those with serially ranking-UP from weak levels for facial acneiform rash induced by EGFRI. MethodsPts with RAS wt mCRC were enrolled in the first registration. All pts received pre-emptive therapy with oral minocycline 100 or 200mg/day and heparinoid moisturizer from the initiation of EGFRIs. Enrolled pts who developed facial acneiform rash within 8 weeks were randomized either to ranking-UP group (UP group) or ranking-DOWN group (DOWN group) (second registration) using minimization method for balancing institution, type of EGFRIs, and sex. Primary endpoint was incidence of Grade2 (moderate) or higher facial acneiform rash during 8 weeks after randomization. Results172 RAS wt mCRC pts, of whom 22 pts and 84 pts received cetuximab and panitumumab, respectively, were enrolled and 106 pts were randomized. There was no significant difference in the incidence of Grade 2 ≧ facial acneiform rash between UP group (18 times) and DOWN group (20 times) (stratified Wilcoxon’s rank sum test, one-sided: p=0.86221). As for secondary end points, proportion of Grade3 or higher facial acneiform rash was 13.2% for UP group and 11.3% for DOWN group, showing no significant difference between the groups (Fisher’s exact test: p=1.0000). There was no problem of safety concern in both groups. ConclusionsTopical corticosteroids ranking UP from weak levels was confirmed to be standard therapy for the management of facial acneiform rash in pts with RAS wt mCRC. It would follow that minocycline and heparinoid moisturizer have a prophylactic efficacy while topical corticosteroids have a therapeutic efficacy for facial acneiform rash. Clinical trial identificationUMIN000024113. Legal entity responsible for the studyFAEISS Study Group. FundingAMED. DisclosureN. Yamazaki: Honoraria (self), Research grant / Funding (institution): Takarabio; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Ono; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): MSD; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis. K. Kikuchi: Research grant / Funding (institution): POLA; Research grant / Funding (institution): Maruho. H. Fukuda: Honoraria (self): Taiho/chugai; Research grant / Funding (self): National Cancer Center. T. Hamaguchi: Honoraria (self), Research grant / Funding (institution): Ono; Honoraria (self): Takeda; Honoraria (self): Bayer. N. Boku: Honoraria (self), Research grant / Funding (institution): Taiho; Honoraria (self), Research grant / Funding (institution): Ono; Honoraria (self), Research grant / Funding (institution): Bristol-Myers Squibb. T. Takenouchi: Speaker Bureau / Expert testimony: Ono; Speaker Bureau / Expert testimony: MSD; Speaker Bureau / Expert testimony: Novartis . T. Nishina: Honoraria (self), Research grant / Funding (institution): Taiho; Honoraria (self), Research grant / Funding (institution): Chugai; Honoraria (self), Research grant / Funding (institution): Merck Serono. S. Yoshikawa: Honoraria (self): Ono; Honoraria (self): Novertis; Honoraria (self): Bristol-Myers Squibb. K. Yamazaki: Honoraria (self): Chugai; Honoraria (self): Daiichi Sankyo. M. Takahashi: Speaker Bureau / Expert testimony, Research grant / Funding (institution): Ono; Speaker Bureau / Expert testimony: Daiichi Sankyo. T. Masuishi: Honoraria (self): Taiho; Honoraria (self): Merck Serono; Honoraria (self): Yakult Honsha. Y. Kiyohara: Honoraria (self), Research grant / Funding (institution): MSD; Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Ono; Honoraria (self), Research grant / Funding (institution): BMS. All other authors have declared no conflicts of interest.

615P - Phase II trial of trametinib (T) and panitumumab (Pmab) in RAS/RAF wild type (wt) metastatic colorectal cancer (mCRC)

615P - Phase II trial of trametinib (T) and panitumumab (Pmab) in RAS/RAF wild type (wt) metastatic colorectal cancer (mCRC)

Update on phase II trial of cetuximab plus S-1/oxaliplatin (SOX) for metastatic colorectal cancer (mCRC): JACCRO CC-06

Abstract Background We have determined the recommended dose of weekly cetuximab (cet) plus SOX (oxaliplatin 130mg/m2 on day 1 and S-1 40 mg/m2 twice daily on days 1-14, every 3 weeks) regimen and showed that it was an effective and tolerable 1st-line regimen in the JACCRO CC-06 trial for KRAS exon2 wild-type (wt) and EGFR-expressing mCRC [J Clin Oncol 2014 32:3_suppl, 571]. However, the follow-up time was short; therefore, the detailed data was missing. Methods The primary endpoint of phase II part was objective response rate (ORR). Progression-free survival (PFS), overall survival (OS), early tumor shrinkage (ETS) at 8 weeks after treatment, depth of response (DpR), and safety were secondary endpoints. The tumor shrinkage was evaluated every 8 weeks by the external review board. Non-preplanned updated analysis was performed in January 2019. Results 59 pts (median 64-y old, 51% male, 85% PS0, 19% right-side) were enrolled into the phase II part. The median cycle of SOX was 6 (range 1-20) with relative dose intensity (RDI) of 80% for oxaliplatin and 83% for S-1. Cet was administered at median times of 17 (range 1-57), the RDI was 95%. Fifty-two (88%) pts discontinued the protocol therapy due to progression (46%) or toxicity (38%). The ORR was 62.7% (95%CI 50.4-75.1), median PFS was 9.1 months (95%CI 6.2-11.6), and median OS was 32.0 months (95%CI 21.5-44.2). In sub-analyses, pts with ETS had significantly longer PFS and OS compared to those without ETS (9.4 vs 3.8 months and 37.0 vs 22.5 months, respectively). Moreover, ETS rate and DpR were comparable between primary tumor sides (left vs. right, 70% vs 80%, 48% vs 46%, respectively). Exploratory analysis by RAS status indicated that ORR was 76.3%, median PFS was 9.4 months, and median OS was 32.0 months in RAS wt population (n = 38). Conclusions SOX plus cet regimen is active with good tumor shrinkage for KRAS wt mCRC regardless of primary tumor sidedness. Pts who experience ETS may potentially have a favorable prognosis.

591P - Impact of primary tumour side on 3-year survival outcomes of first-line (1L) FOLFOX-4 ± cetuximab in patients with RAS wild-type (wt) metastatic colorectal cancer (mCRC) in the phase III TAILOR trial

BackgroundThe TAILOR trial demonstrated a significant survival advantage with the addition of cetuximab to 1L FOLFOX-4 in patients from China with RAS wt mCRC and met all of its endpoints. We report the impact of primary tumor side on 3-year overall survival (OS) in TAILOR. MethodsTAILOR was a randomized, open-label, multicenter, phase 3 trial that evaluated 1L cetuximab + FOLFOX-4 vs FOLFOX-4 in patients from China with RAS wt mCRC. The primary endpoint was progression-free survival time, as determined by independent review committee; secondary endpoints included OS time, ORR, and safety/tolerability. Tumors were categorized in evaluable patients as left (L)-sided (splenic flexure, descending colon, sigmoid colon, and rectum) or right (R)-sided (appendix, cecum, ascending colon, hepatic flexure, and transverse colon). ResultsOf the 393 treated patients with RAS wt mCRC, median follow-up time was 57.0 months (range, 1.5–75.5), and 391 patients were evaluable for tumor side. Median OS and 3-year OS rates by primary tumor side are summarized in the table.Table591PTablePrimary tumor side: LPrimary tumor side: RCetuximab + FOLFOX-4 (n=146)FOLFOX-4 (n=162)Cetuximab + FOLFOX-4 (n=45)FOLFOX-4 (n=38)Median OS (95% CI), months22.0 (18.7–25.5)18.3 (15.2–20.2)11.5 (7.5–20.4)9.4 (7.5–18.8)3-year OS rate (95% CI), %32 (25–40)16 (11–22)23 (11–38)14 (5–28) ConclusionsThis updated analysis at>4.5 years’ median follow-up is consistent with previous results from the TAILOR study, which showed improved OS with the addition of cetuximab to FOLFOX-4. Indeed, the 3-year OS rate was doubled in patients with L-sided mCRC and > 1.5 times higher in those with R-sided disease, showing that patients with mCRC might benefit from treatment with cetuximab irrespective of primary tumor side. Overall, these results confirm cetuximab in combination with FOLFOX-4 as an effective standard-of-care 1L therapy for patients with RAS wt mCRC. Clinical trial identificationNCT01228734; EMR62202-057. Editorial acknowledgementMedical writing assistance (funded by Merck Healthcare KGaA, Darmstadt, Germany) was provided by Marjorie Rummelt, PhD, of ClinicalThinking, Inc, Hamilton, NJ, USA. Legal entity responsible for the studyMerck Healthcare KGaA. FundingMerck Healthcare KGaA. DisclosureJ. Nippgen: Full / Part-time employment: Merck Serono. W. Chen: Full / Part-time employment: Merck Serono. J. Li: Research grant / Funding (institution): Merck; Research grant / Funding (institution): Roche. All other authors have declared no conflicts of interest.

527PD - Trastuzumab and tucatinib for the treatment of HER2 amplified metastatic colorectal cancer (mCRC): Initial results from the MOUNTAINEER trial

BackgroundHER2 (ERBB2) amplification is present in 5-10% of patients (pts) with KRAS and NRAS (RAS) WT mCRC. Tucatinib (Seattle Genetics, Bothell, WA) is a potent, highly selective oral tyrosine kinase inhibitor of the HER2 receptor. In patient-derived xenograft models of HER2+ mCRC, the combination of tucatinib and trastuzumab showed significantly greater antitumor activity compared with tucatinib or trastuzumab alone. Here we present initial results from a trial of tucatinib and trastuzumab in pts with HER2 amplified mCRC. MethodsMOUNTAINEER is a multicenter open-label single-arm phase II trial. Pts with RAS WT mCRC had HER2 amplification/overexpression by NGS, FISH, or IHC (3+ or 2+ and amplified by FISH). Prior treatment with 5FU, oxaliplatin, irinotecan, and an anti-VEGF antibody was required. Prior anti-HER2 therapy was excluded. Pts received tucatinib 300mg PO bid and standard doses of trastuzumab (IV, Q3 weeks). The primary endpoint was objective response rate (ORR) per RECIST v1.1. Using a 2-Stage Fleming design, this study compared ORR of 20% (null) vs 40% (alt) in 10 or 25 evaluable pts (1-sided α=0.11; β=.16). At least 8 responses were needed to meet the primary efficacy endpoint. ResultsAs of 26 April 2019, 26 pts enrolled, and 22 pts completed ≥1 evaluation of response. 16/26 pts (62%) were male. Median age= 52.5 (range 24-70). The primary tumor site of origin included right colon (N=4), left colon/rectum (N=17), transverse colon (N=3), and overlapping (N=2). Among 22 evaluable pts, ORR= 55% (CR/PR= 12; SD=5; PD=5). Clinical benefit rate (CR+PR+SD≥4 months) = 64%. At a median follow-up of 10.6 months, median PFS= 6.2 months (95% CI 3.5-NE). Median OS=17.3 months (95% CI 12.3-NE). Median duration of response has not been reached. There were 2 (9%) grade 3 treatment-related adverse events (TRAEs) and no grade 4/5 TRAEs. The most common TRAEs were AST elevation (48%; all G1), ALT elevation (30%; all G1), and diarrhea (26%; G1/G2/G3=4%/17%/4%). ConclusionsThe combination of tucatinib and trastuzumab is well tolerated and has met its primary efficacy endpoint. Based on these results, further expansion of the MOUNTAINEER trial in pts with HER2 amplified mCRC is justified. Updated results will be presented. Clinical trial identificationNCT03043313. Legal entity responsible for the studyAcademic and Community Cancer Research United. FundingSeattle Genetics. DisclosureJ.H. Strickler: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Amgen; Honoraria (self), Advisory / Consultancy: Bayer; Honoraria (self): Chugai; Advisory / Consultancy, Research grant / Funding (institution): Genentech/Roche; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Seattle Genetics; Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options, Non-remunerated activity/ies: OncoMed; Advisory / Consultancy: Celgene; Advisory / Consultancy: Proteus Digital Health; Advisory / Consultancy: Chengdu Kanghong Biotechnology Ltd; Research grant / Funding (institution): Exelixis; Research grant / Funding (institution): Sanofi; Research grant / Funding (institution): Nektar; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): Macrogenics; Research grant / Funding (institution): Leap Therapeutics. A. Cercek: Advisory / Consultancy: Bayer; Advisory / Consultancy: Proteus; Research grant / Funding (institution): Seattle Genetics. C. Wu: Research grant / Funding (institution): Vaccinex; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Lycera; Research grant / Funding (institution): Seattle Genetics; Research grant / Funding (institution): Symphogen; Research grant / Funding (institution): FLX Bio. J. Hubbard: Research grant / Funding (institution): Seattle Genetics; Honoraria (institution), Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Treos Bio; Research grant / Funding (institution): Taiho; Research grant / Funding (institution): Senhwa pharmaceuticals. N. Kemeny: Research grant / Funding (institution): Amgen. P.M. Boland: Research grant / Funding (institution): Merck; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Hemispherx; Research grant / Funding (institution): Boston Biomedical; Research grant / Funding (institution): Isofol Medical; Research grant / Funding (institution), Travel / Accommodation / Expenses: Ipsen; Research grant / Funding (institution): Athenex; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Clinical Genomics. K. Ng: Advisory / Consultancy, Research grant / Funding (institution): Genentech; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Tarrex Biopharma; Advisory / Consultancy: Bayer; Advisory / Consultancy, Research grant / Funding (institution): Seattle Genetics; Research grant / Funding (institution): Gilead Sciences; Research grant / Funding (institution): Trovagene; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Pharmavite; Research grant / Funding (institution): Consano. T. Bekaii-Saab: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Seattle Genetics. All other authors have declared no conflicts of interest.

Dynamic monitoring of HER2 amplification in circulating DNA of patients with metastatic colorectal cancer treated with cetuximab.

Cetuximab (CTX) has been used to treat metastatic colorectal cancer (mCRC) with wild-type (wt) RAS and BRAF genes. Meanwhile HER2 amplification reportedly denoted CTX-resistant mCRC tumors. We investigated whether monitoring of HER2 amplification in circulating DNA allowed early detection of mCRC progression and CTX resistance. We analyzed HER2 amplification in circulating DNA at 8-week intervals using ddPCR from 36 patients with RASwt/BRAFwt mCRC, who progressed after CTX treatments between July 2015 and January 2018. Of the 36 patients, 5 (13.9%) exhibited dynamic fluctuations of HER2 amplification in plasma in the course of CTX treatment, of whom 2 were positive for HER2 amplification in matched tumor specimens at baseline (per FISH). All 5 primary sites were left side: 3 rectums and 2 descending colon. HER2 ratio fluctuations in circulating DNA not only reflected changes in tumor volume, but their obvious increases presaged CT-documented progress by an average lead time of 2months. Interestingly, progression-free survival did not significantly differ between these 5 patients and those without HER2 amplification (HR 1.06, 95% CI 0.40-2.77, P = 0.909). Plasma HER2 amplification detected by ddPCR changed over time and predicted resistance to CTX, by an average lead time of 2months. Further study is needed to validate our findings.

How to Deal with Second Line Dilemma in Metastatic Colorectal Cancer? A Systematic Review and Meta-Analysis

Monoclonal antibodies targeting epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) have demonstrated efficacy with chemotherapy (CT) as second line treatment for metastatic colorectal cancer (mCRC). The right sequence of the treatments in all RAS (KRAS/NRAS) wild type (wt) patients has not precisely defined. We evaluated the impact of aforementioned targeted therapies in second line setting, analyzing efficacy and safety data from phase III clinical trials. We performed both direct and indirect comparisons between anti-EGFR and anti-VEGF. Outcomes included disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and G3-G5 toxicities. Our results showed significantly improved OS (HR 0.83, 95% CI 0.72–0.94) and DCR (HR 1.27, 95% CI 1.04–1.54) favouring anti-VEGF combinations in overall population; no statistically significant differences in all RAS wt patients was observed (HR 0.87, 95% CI 0.70–1.09). Anti-EGFR combinations significantly increased ORR in all patients (RR 0.54, 95% CI 0.31–0.96), showing a trend also in all RAS wt patients (RR 0.63, 95% CI 0.48–0.83). No significant difference in PFS and DCR all RAS was registered. Our results provided for the first time a strong rationale to manage both targeted agents in second line setting.

Abstract LB-265: ARAF activates RAS by antagonizing its interaction with NF1

Abstract ARAF, BRAF and CRAF protein kinases are RAS effectors required for activation of ERK signaling. ARAF has relatively modest kinase activity and its biological functions are largely unknown. Here, we show that ectopic expression of ARAF but not B- or CRAF, leads to induction of RAS activation in cells with WT RAS. This phenomenon requires the binding of ARAF to RAS but not its kinase activity. ARAF was found to reduce inactivation of RAS by antagonizing its binding to the major RAS-GAP NF1. By this mechanism, the abundance of ARAF in cells determines cellular RAS-GTP levels, the dynamics of RTK-induced RAS activation and the biological consequences of RAS signaling. We find that a certain expression level of ARAF is required for growth factors induced proliferating or neural differentiation in PC12 cells. In RTK-dependent tumor cells, knocking down of ARAF leads to a reduction of RAS-GTP, thus impairs RAS signaling and the tumor growth. Furthermore, by investigating the tumor samples from MSK patients who are diagnosed with progressing disease after EGFR inhibitor treatment, we find the up-regulation of ARAF (including the ARAF gene amplification) is associated with EGFR inhibitor resistance in the lung cancer patients. We confirmed in cell line and xenograft models that a 5-fold elevation of ARAF expression is sufficient to eliminate RAS inhibition by EGFR inhibition and to cause resistance. Resistance in these cells could be overcome by combining EGFR and SHP2 inhibitors. The latter prevents EGFR activation of RAS-GTP exchange and thus would be expected with weaking the effects of NF1 inhibition by ARAF. Taken together, our work shows that ARAF directly activates RAS by antagonizing its interaction with NF1 and thus affects the duration and cellular response of growth factor activation and sensitivity of tumors to pharmacological inhibition of RTK-RAS signaling. Citation Format: Wenjing Su, Rona Yaeger, Na Na, Jaclyn Hechtman, Viktoriya Paroder, Sandra Misale, Sae-Won Han, Omar I. Abdel-Wahab, Frank McCormick, Neal Rosen, Zhan Yao. ARAF activates RAS by antagonizing its interaction with NF1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-265.

Abstract 956: An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development

Abstract The RAS gene family is among the most commonly mutated genes within cancer, but little progress has been made in successfully targeting RAS mutations. Targeting binding partners of mutated RAS presents as a promising alternative therapeutic strategy. With the goal of uncovering novel interactors of RAS, we recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the KRAS through its Switch II domain. In order to assess the role of AGO2 in KRASG12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development that failed to progress to late stage PanINs, pancreatic ductal adenocarcinoma (PDAC), and metastatic disease. This resulted in a dramatic increase in the survival of mice with AGO2 ablation. Unlike the PanINs in AGO2 sufficient mice, the early PanIN lesions with AGO2 ablation showed staining for the senescence associated beta galactosidase activity, suggesting that AGO2 loss induces oncogene induced senescence. To extend these observations and explore the role of AGO2 interaction with mutant forms of HRAS and NRAS proteins, we performed co-IP of AGO2 with RAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2 in T24 cells (HRASG12V) and SK-MEL-2 cells (NRASQ61H), respectively. In T24 cells, AGO2 knockdown led to the senescent phenotype and was accompanied with changes in the EGFR-RAS signaling axis, similar to that observed in the PanINs of the mice with AGO2 loss. In this cell line model, AGO2 loss reduced mutant HRAS expression and increased wild type RAS activity. These signaling effects were also consistent with our observation that AGO2 loss increased RAS activation in the mouse embryonic fibroblast (MEF) model. Together with our previous work with mutant KRAS dependent cells, these data suggest that 1) AGO2-wild type RAS binding prevents RAS activation and 2) mutant RAS-AGO2 association regulates oncogenic RAS levels in cell line models. Studies on the mouse model and the close proximity of RAS and AGO2 with EGFR also furthered our understanding of the RAS-AGO2 interaction. Using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupts the interaction between WT RAS and AGO2. However, the mutant KRAS-AGO2 interaction was recalcitrant to EGFR regulation. This provides the first instance of a nucleotide dependent association of RAS and AGO2 and sheds light on the dynamic nature of the RAS-AGO2 interaction. Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Seema Chugh, Ingrid J. Apel, Malay Mody, Anudeeta Gautam, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 956.

Retrospective analysis of overall survival (OS) by subsequent therapy in patients (pts) with RAS wild-type (wt) metastatic colorectal cancer (mCRC) receiving irinotecan ± cetuximab in the EPIC study.

3580 Background: The multicenter, open-label, randomized, phase 3 EPIC study (EMR 062202-025) investigated cetuximab + irinotecan vs irinotecan as a second-line (2L) therapy in pts with EGFR-detectable mCRC. A retrospective analysis in the EPIC RAS wt population showed improved overall response rate (29.4% vs 5.0%) and progression-free survival (5.4 vs 2.6 mo) upon the addition of cetuximab to irinotecan. Median OS (mOS) was similar in both treatment arms, possibly due to differences in subsequent therapies. We present OS by post-study therapy in the RAS wt population. Methods: 1298 RAS-unselected pts were enrolled from May 2003 to February 2006. The primary endpoint was OS. RAS status was determined retrospectively in 2018 from existing DNA samples using BEAMing technology; wt status was defined as having a sum of mutated RAS allele frequencies of ≤5%, with all relevant alleles being analyzable. Results: Among the 452 pts with RAS wt mCRC, 231 received cetuximab + irinotecan and 221 received irinotecan. Baseline characteristics were similar in both arms. OS data by post-study therapy are summarized in the Table. No new or unexpected safety signals were observed. Conclusions: Post-study cetuximab was associated with improved OS in both treatment arms compared with post-study therapy without cetuximab and no subsequent therapy, suggesting that cetuximab-based therapy may be suitable as a standard treatment for pts with RAS wt mCRC in the rechallenge setting. Study limitations include a potential bias due to the differences in proportion of subsequent therapies with and without cetuximab between arms (almost 50% of pts in the irinotecan arm received post-study cetuximab) as well as the likelihood for pts who live longer to receive cetuximab in any subsequent therapy line. [Table: see text]

Randomized phase II trial of adjuvant hepatic arterial infusion (HAI) + systemic FOLFIRI +/- panitumumab (Pmab) in patients with resected RAS wild type colorectal cancer hepatic metastases (CRLM).

3555 Background: HAI therapy has improved recurrence free (RF) survival in several randomized trials after resection of CRLM. The purpose of this trial was to determine whether systemic Pmab added to adjuvant HAI + FOLFIRI in RAS WT pts increases 15 months RF survival (RFS) after liver resection. Secondary endpoints are evaluation of overall survival, toxicity, and predictive biomarkers. Methods: RAS WT pts with resected liver mets were randomized to HAI + SYS (+/-) Pmab after stratification by clinical risk score (≥ 3 or < 3) and previous chemotherapy (Y/N). For a particular arm, if 24 or more patients (pts) were alive and RF at 15 months, the regimen in that arm would be considered worthy of further investigation. The initial dose of HAI FUDR 0.12mg/Kg + dexamethasone was infused over the first 2 weeks of a 5-week cycle. Systemic chemotherapy was delivered on days 15 and 29 (irinotecan 125 mg/m2, LV 400 mg/ m2, 5FU 1000 mg/m2 48-hour continuous infusion and +/- Pmab 6mg/kg). Patient characteristics were compared between arms using Fisher’s exact test and Wilcoxon rank-sum test. Survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test. Results: After randomization of 75 pts, the arm receiving + Pmab met the decision rule of having ≥ 24 patients alive and RF at 15 months. The two arms had similar pt characteristics and toxicity, with the exception of Pmab related rash (Table). The 15-month RFS is 79% and 67% in +/- Pmab arms, respectively. With a median follow-up of 45 months, 3-year RFS is 65% [CL 0.45-0.78] and 42% [CL 0.24-0.57], and 3-year survival is 96% and 90% in +/-Pmab arms, respectively. Conclusions: In this trial, the addition of Pmab to HAI and SYS showed promising activity without increase in biliary toxicity and should be further investigated in a larger study. Predictive biomarkers will be presented. Clinical trial information: NCT01312857. [Table: see text]

Effect of patient age on efficacy of FOLFIRI plus cetuximab vs bevacizumab in 1st-line treatment of metastatic colorectal cancer: An analysis of FIRE-3 (AIO KRK 0306).

3541 Background: FIRE-3 compared 1st-line therapy with FOLFIRI plus either cetuximab or bevacizumab in 592 KRAS exon 2 wt mCRC patients. The subgroup of extended RASwt patients consisted of 400 patients. The median age of patients treated in FIRE-3 was 64 years. Analyses of efficacy of doublet-chemotherapy in patients >70 years have shown inconsistent results. Methods: In this exploratory analysis, patients were grouped into cohorts with 65 years and 70 years as a cut-off for age-related analysis of the FIRE-3 study population. ORR was compared using Fisher´s exact test, Survival analyses were done using Kaplan-Meier estimation and median survival times were compared using log-rank testing. Results: Within the RAS wt population, patients older than 70 years had a significantly shorter OS when compared to patients ≤70 years of age in both arms (p= 0.02 for cetuximab arm, p= 0.02 for bevacizumab arm). Patients ≤65 years and ≤70 years had a significantly longer OS when treated with FOLFIRI plus cetuximab compared to FOLFIRI plus bevacizumab (p= 0.01 and 0.02 respectively). The OS benefit of cetuximab treated patients compared to bevacizumab treated patients was consistent for patients ≤65 years (p=0.005) and patients ≤70 years (p= 0.009) of age. In patients older than 70 years, however, comparable efficacy of bevacizumab and cetuximab was observed. This effect of age on outcome appears to be affected by sidedness. RAS wt population (n=400). Conclusions: In the overall RAS wt population, younger patients have a significant OS benefit when treated with FOLFIRI plus cetuximab compared to FOLFIRI plus bevacizumab, while this was not the case in patients older than 70 years.[Table: see text]

Molecular analysis of advanced colorectal cancer in a patient cohort of Hispanics, African Americans, and Caucasians within a diverse single institution.

e15121 Background: CRC survival and treatment response differences have been noted across racial and ethnic groups, particularly between Hispanics (HA), non-Hispanic AA (AA), and non-Hispanic Caucasian (CA). We examined if there are differences in molecular drivers between these groups that could explain such differences in our diverse medical center. Methods: We retrospectively examined 361 HA, AA, CA patients at Montefiore Medical Center diagnosed with metastatic CRC (mCRC) from 2007 to 2018. Mismatch repair proficiency (MMR-P), deficiency (MMR-D) was determined by immunohistochemistry. KRAS, NRAS, BRAF were determined by polymerase chain reaction or next generation sequencing. Mutations were then correlated with overall survival (OS). Results: Across subgroups, there was no differences in MMR, KRAS, NRAS, BRAF incidence (Table). Using a univariate analysis, the median survival for RAS mutated (M) patients was 33 months (ms), compared to 69 ms for RAS wild-type (WT, p = 0.01). The median OS for RAS mutated HA patients was 41 ms, compared to 41 ms and 33 ms for CA and AA respectively (p = 0.14). There was also no statistically significant difference between median OS for RAS WT HA patients (69 ms), compared to AA patients (51 ms) and CA (not reached, p = 0.75). Conclusions: In our single institution study, we found no difference in incidence of RAS (KRAS and NRAS), BRAF, MMR amongst HA, AA, and CA. Delivery of uniform care may account for similar OS outcomes in our patient population. RAS WT and M disease had improved OS compared to previously established rates in the literature. Additional studies are needed to evaluate known differences in treatment response and survival in minority populations.[Table: see text]

Association of microRNA-21 with efficacy of cetuximab in RAS wild-type patients in the FIRE-3 study (AIO KRK-0306) and microRNA-21’s influence on gene expression in the EGFR signaling pathway.

3593 Background: FIRE-3 compared first-line therapy with FOLFIRI plus cetuximab (cet) or bevacizumab (bev) in KRAS exon 2 wild-type (wt) patients with metastatic colorectal cancer. Recent analyses showed mircoRNA-21 (miR-21) expression level may be a predictive biomarker for anti-EGFR-therapy raising the question whether miR-21 influences gene expression in the EGFR signaling pathway. Methods: Reverse-transcription quantitative polymerase chain reaction assay identified quantitative miR-21 expression. Median expression was defined as a threshold value to discriminate FIRE-3 population into miR-21 low and high groups. Differential gene expression based on additional mRNA microarray data (Almac Inc, Xcel Array) was calculated by linear models adjusted for multiple testing followed by single sample gene set enrichment analysis (ssGSEA) to compare differentially enriched hallmarks of cancer gene sets. Overall response rate (ORR) was compared using Fisher´s exact test. Median progression-free (PFS) and overall survival (OS) were analyzed using Kaplan-Meier estimation and log-rank test. Results: 333 RAS wt patients provided material for miR-21 expression analysis. In these patients, low miR-21 expression was associated with higher ORR (80.0% vs. 57.9%; p = 0.005) and longer OS (35.8 months (mo) vs. 25.9 mo; p = 0.005) when cet vs bev was added to FOLFIRI. High miR-21 expression was associated with comparable ORR (74.6% vs. 64.0%; p = 0.21) and OS (24.5 mo vs. 23.8 mo; p = 0.4). There was no significant difference in PFS in either group. By comparing miR-21 low and high groups using normalized mRNA microarray data, 538 genes were found to be significantly differentially expressed in RAS wt patients after adjustment for multiple testing. Including data from the two groups into ssGSEA yielded 23 hallmark of cancer gene sets that were significantly differentially enriched; among them, KRAS-signaling showed higher enrichment in the miR-21 high group (adjusted p = 2.09 E-13). Conclusions: MiR-21 expression level might be a predictive biomarker for anti-EGFR-therapy by modulating KRAS signaling in FIRE-3 patients.

An evaluation of RAS testing and survival among mCRC patients in the United States.

e15144 Background: The status of mutations in the rat sarcoma viral oncogene homolog ( RAS) family of genes, which includes Kirsten RAS ( KRAS) and Neuroblastoma RAS ( NRAS) predicts response to anti-EGFR therapies in metastatic colorectal cancer (mCRC) patients. Current treatment guidelines recommend all mCRC patients to receive RAS testing prior to initiating treatment with an anti-EGFR agent. This study sought to establish estimates of RAS testing among mCRC patients in the US prior to initiation of first and third lines of treatment, and to describe median survival by treatment groups. Methods: Data from the Oncology Services Comprehensive Electronic Records (OSCER) 2.0 dataset were utilized. Patients diagnosed with mCRC between January 1, 2011 and July 31, 2017 were eligible. Patients were followed for up to 1 year, or until death, following their mCRC diagnosis. Results: A total of 17,387 mCRC patients were included in the analysis, among which 69% were RAS tested and 31% were never tested. Among the RAS tested patients, 23% were tested prior to their mCRC diagnosis 60% received RAS testing following mCRC diagnosis but prior to first line of treatment, 3% were tested following first line treatment but prior to third line, and the remaining 14% were tested following third line. Demographic variables did not differ between tested and untested groups, overall and by line of treatment. The overall median survival of RAS WT patients was 31.1 months (95% CI: 30.0, 32.4). MCRC patients who were not RAS tested had a lower median survival of 20.7 months (95% CI: 19.1, 22.5). Conclusions: The timing of when patients are being tested varies greatly. While nearly 70% of patients had a RAS test, approximately 30% failed to be tested, identifying a large gap in testing practices. Universal RAS testing provides patients and their physicians with knowledge of their anti-EGFR treatment options and may result in improved survival.

How to deal with second line dilemma in metastatic colorectal cancer? A systematic review and meta-analysis.

e15006 Background: Monoclonal antibodies targeting epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) have demonstrated efficacy in combination with chemotherapy as second line for metastatic colorectal cancer (mCRC). However, there is still a paucity of evidence or guidelines suggesting the right sequential treatment in all RAS (KRAS/NRAS) wild type(wt)mCRC. Therefore, we aimed to evaluate the impact of these targeted therapies by reviewing literature data. Methods: We used Cochrane, EMBASE and Medline databases to select phase III clinical trials containing efficacy and safety data about chemotherapy (CT) or CT + targeted agents combination (Anti-VEGF and Anti-EGFR) in second line mCRC setting. We performed direct comparisons to obtain pooled data for anti-VEGF + CT versus CT and anti-EGFR +CT versus CT comparisons. Then we performed indirect comparisons between anti-EGFR and Anti-VEGF. Outcomes were disease control rate (DCR), response rate (RR), progression-free survival (PFS), overall survival (OS) and most common G3-G5 toxicities. Results: Eight eligible RCTs (6793 pts) were included: 5 studies compared anti-VEGF + CT and 3 anti-EGFR + CT combinations to CT. After direct comparisons, pooled indirect results showed significantly improved OS (HR 0.83, 95% CI 0.72–0.94) and DCR (HR 1.27, 95% CI 1.04–1.54) favouring anti-VEGF combinations in overall population; however, no statistically significant differences in all RAS wt patients was observed (HR 0.87, 95% CI 0.70–1.09). Additionally, anti-EGFR combinations significantly increased ORR in all patients (RR 0.54, 95% CI 0.31–0.96), showing a trend in all RAS wt patients (RR 0.63, 95% CI 0.48–0.83) too. Furthermore, no significant difference in PFS and DCR all RAS was registered. Anti-VEGF combination significantly increased Only a significant asthenia difference (RR 1.34, 95% CI 1.03–1.75) was registered. Conclusions: At our knowledge, our indirect comparisons between anti-VEGF and anti-EGFR combinations showed for the first time better OS and DCR for anti-VEGF combinations, whereas better RR is observed for anti-EGFR combinatory regimens, defining a role of both targeted agents in second line mCRC setting.

Genomic alterations after EGFR blockade in patients with RAS wild-type metastatic colorectal cancer: Combined tissue and blood-based analysis from SCRUM-Japan GI-SCREEN and GOZILA.

3528 Background: Anti-EGFR therapy (tx) in RAS wild-type (wt) metastatic colorectal cancer (mCRC) induces resistance through acquired genomic alterations. We aimed to define such alterations in Nationwide Cancer Genome Screening Project tissue and blood specimens, using next generation sequencing (NGS). Methods: Tumor specimens in patients (pts) with RAS wt mCRC were obtained from SCRUM-Japan GI-SCREEN (tissue) and GOZILA (circulating tumor DNA [ctDNA] from blood). Genomic alterations were compared using the Oncomine Comprehensive Assay (SCRUM) and Guardant360 (GOZILA), before anti-EGFR tx and after progression. Results: 373 total actionable alterations were identified in 71 pts with available matched tissue and ctDNA; 255 (68%) were acquired after anti-EGFR tx progression. Frequently seen acquired oncogenic alterations included KRAS mutations (27%) and amplifications (amps) of EGFR (41%), CDK6 (24%), BRAF (20%), MYC (17%), MET (14%), PIK3CA (11%), FGFR1 (11%), and KRAS (10%). Fusions of RET, ALK, and FGFR3 were newly acquired in 1-4%. Acquired alterations co-arose in multiple pathways, including the cell cycle, PI3K-AKT, and MAPK, although 29% of pts had none. Acquired mutations were less frequently clonal versus primary mutations (p<0.0001), but clonal acquired mutations were seen in several oncogenes, including EGFR, KRAS, and PIK3CA. A subset of acquired KRAS, MET, CCND2, and EGFR amps had high (>7) adjusted plasma copy numbers (ApCN). Acquired ERBB2 amps were identified in 3 pts (4%) with a median ApCN of 4, one of whom (ApCN=4.2), treated with dual HER2 blockade, progressed after 2 cycles. Conclusions: Our integrated analysis revealed that anti-EGFR tx of pts with RAS WT mCRC led to acquired genomic alterations in multiple oncogenic pathways. Although most acquired alterations were subclonal, a subset of oncogenic alterations had relatively high clonality and ApCN, suggesting potential targets for overcoming acquired resistance to anti-EGFR tx. Early progression in a pt with an ApCN of 4.2 suggests low-level/subclonal acquired alterations may not be effective treatment targets.