NRAS Mutations Research Articles

Comprehensive analysis of gene mutations and mismatch repair in Chinese colorectal cancer patients.

RAS, BRAF, and mismatch repair (MMR)/microsatellite instability (MSI) are crucial biomarkers recommended by clinical practice guidelines for colorectal cancer (CRC). However, their characteristics and influencing factors in Chinese patients have not been thoroughly described. To analyze the clinicopathological features of KRAS, NRAS, BRAF, and PIK3CA mutations and the DNA MMR status in CRC. We enrolled 2271 Chinese CRC patients at the China-Japan Friendship Hospital. MMR proteins were tested using immunohistochemical analysis, and the KRAS/NRAS/BRAF/PIK3CA mutations were determined using quantitative polymerase chain reaction. Microsatellite status was determined using an MSI detection kit. Statistical analyses were conducted using SPSS software and logistic regression. The KRAS, NRAS, BRAF, and PIK3CA mutations were detected in 44.6%, 3.4%, 3.7%, and 3.9% of CRC patients, respectively. KRAS mutations were more likely to occur in patients with moderate-to-high differentiation. BRAF mutations were more likely to occur in patients with right-sided CRC, poorly differentiated, or no perineural invasion. Deficient MMR (dMMR) was detected in 7.9% of all patients and 16.8% of those with mucinous adenocarcinomas. KRAS, NRAS, BRAF, and PIK3CA mutations were detected in 29.6%, 1.1%, 8.1%, and 22.3% of patients with dMMR, respectively. The dMMR was more likely to occur in patients with a family history of CRC, aged < 50 years, right-sided CRC, poorly differentiated histology, no perineural invasion, and with carcinoma in situ, stage I, or stage II tumors. This study analyzed the molecular profiles of KRAS, NRAS, BRAF, PIK3CA, and MMR/MSI in CRC, identifying key influencing factors, with implications for clinical management of CRC.

Abstract IA001: Exploiting pathway activation as a new form of synthetic lethality

Abstract Targeting activated oncogenes is an effective treatment strategy across many cancers now including therapeutics targeting KRAS. Genetic events including DNA damage deficiencies and tumor suppressor mutations require alternative strategies and the concept of synthetic lethality has been applied to these alterations. PARPi inhibitors were a founding member of this class of therapeutics demonstrating enhanced activity in the setting of BRCA-deficiency. By applying large-scale loss-of-function approaches1 we and other discovered the vulnerability to PRMT5 and WRN inhibition imposed by co-deletions of MTAP and CDKN2A, and the MSI+ genotype respectively2,3. Such inhibitors are now in clinical trials. To explore the potential for paralogous genes to act as synthetic lethal gene pairs we enacted dual knockout screens. These early efforts led to the discovery of DUSP4/6 paralog dependence in the setting of BRAF and NRAS mutations in melanoma4. Intriguingly the loss of DUSP4/6 impaired cancer cell viability through the increased activation of ERK highlighting the susceptibility of cancers to pathway activation in addition to the more common sensitivity to pathway inhibition. This inappropriate activation of the ERK signaling pathway, the conflict between EGFR and KRAS activation, the synthetic lethality enacted by TRIM8 knockouts, and the effects of AR agonists on prostate cancer viability, points to a wider than expected vulnerability of cancer to inappropriate gene activation. To systematically identify context-specific gene activation induced lethalities in cancer, we developed methods for enacting gain-of-function perturbations across ∼500 barcoded cancer cell lines. With this approach, we queried the pan-cancer vulnerability landscape upon activating 10 key cancer pathway revealing activation dependencies in MAPK and PI3K pathways. Notably, we discovered novel pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct β-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. These latter discoveries paradoxically point to the residual activity of the APC ubiquitin-ligase complex as a target in APC-mutant CRC5. 1. McDonald, E. R., 3rd et al. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577-592.e10 (2017). 2. Mavrakis, K. J. et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science 351, 1208–1213 (2016). 3. Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551–556 (2019). 4. Ito, T. et al. Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers. Nat. Genet. 53, 1664–1672 (2021). 5. Chang, L. et al. Systematic profiling of conditional pathway activation identifies contextdependent synthetic lethalities. Nat. Genet. 55, 1709–1720 (2023). Citation Format: William R. Sellers. Exploiting pathway activation as a new form of synthetic lethality [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA001.

Development of custom amplicon-based test systems for detecting KRAS, NRAS, and BRAF gene mutations in colorectal cancer using next-generation sequencing (NGS).

e15061 Background: Polymerase chain reaction (PCR)-based mutation diagnostics in colorectal cancer is widely available. However, PCR has limitations in identifying rare mutations and precise mutation coordinates. Targeted sequencing, though promising, often incurs high costs and longer research durations, demanding higher DNA sample quality. Our study demonstrates the benefits of using a customized amplicon test system for diagnosing somatic mutations in tumor tissue, including detecting rare mutations not captured by PCR, requiring less input biomaterial, and similar research costs. Methods: A targeted NGS assay system was developed using region-specific primers for KRAS (exons 2, 3, and 4), NRAS (exons 2, 3, and 4), and BRAF (exon 15). Library preparation involved a three-step PCR process: Target PCR incorporating Illumina overhang adapter sequences, Index PCR introducing multiplexing indices and sequencing adapters, and PCR cleanup using magnetic beads. Sequencing utilized MiSeq v2 reagents with paired 300‐bp reads, generating high-quality, full-length reads. Validation included re-testing known samples. Tumor cell percentages in FFPE specimens were determined, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Data analysis utilized proprietary software. Results: Out of 1234 samples, 1133 were tested, with mutations detected in 685 samples (60.45%). KRAS mutations were found in 548 (48.36%), NRAS mutations in 40 (3.5%), and BRAF mutations in 88 (7.7%) samples. Double mutations were observed in 9 samples (0.79%). Data on rare and double mutations are presented in the table. Conclusions: The developed test system accurately identifies essential mutations in colorectal cancer, including rare and potentially clinically significant variants. The required DNA amount ranged from 10 to 30 ng in 5 μl, nearly ten times less than well-known commercial PCR test systems like COBAS (Roche) and therascreen (QIAGEN). The cost of the study is comparable to PCR with a minimum study duration of 4 days. [Table: see text]

PAS-004: A novel macrocyclic MEK inhibitor to inhibit cancer cell growth in vitro and tumor growth in mouse xenograft studies.

3126 Background: Three MEK inhibitors have been approved to date to treat melanomas driven by RAS pathway mutations and a 4th compound (selumetinib) is approved to treat Neurofibromatosis type 1 (NF1) in pediatric patients. PAS-004 is the first macrocyclic MEK inhibitor in development for solid tumors and NF1. Methods: Ten NRAS mutant cancer cell lines were cultured for 48 hrs in the presence of the MEK inhibitor PAS-004, trametinib, binimetinib (bini) and selumetinib (selu) and cell proliferation was assayed using a Cell Titer Glo luminescence assay. The anti-tumor activity of PAS-004 was compared with selu and bini in two mouse xenograft studies using NRAS mutant human hepatocellular carcinoma (HCC) and lung carcinoma cell lines. Dose response was assessed with treatment initiated when the tumor reached a volume of 150 mm3 and mice treated for 20 days. Body weights and tumor volumes were measured twice weekly. Tumors were excised at the end of the study and pERK inhibition was assessed by Western blot. Terminal plasma was collected for PK analysis. Results: PAS-004 strongly inhibited 5/10 NRAS mutant cancer lines with IC50 values ranging from 0.024 to 0.306 µM. Maximal growth inhibition of &gt;50% was achieved by PAS-004 in 8 cell lines and PAS-004 achieved greater maximal inhibition in 7/10 lines vs selu and bini. PAS-004 inhibition was comparable to trametinib in 5/10 cell lines tested. However, in contrast to trametinib PAS-004 did not reach a plateau at the highest dose tested in three of these lines. In the HCC xenograft study the highest dose of PAS-004 completely prevented tumor growth and the same dose caused a 69% reduction in tumor volume in the lung carcinoma xenograft study. The anti-tumor efficacy of PAS-004, when taken at equivalent doses, was shown to be superior to that of bini and selu. Specifically, PAS-004 at dose levels of 10 mg/kg and 5 mg/kg, once daily, exhibited higher efficacy compared to bini and selu at dose levels of 5 mg/kg and 2.5 mg/kg, when administered twice daily, respectively. No significant differences in body weight loss were seen between the compounds. Conclusions: The novel macrocyclic MEK inhibitorPAS-004 inhibited NRAS mutant tumor growth in mouse xenograft studies with similar activity as the approved MEK inhibitors selu and bini. In vitro, PAS-004 provided greater maximal growth inhibition of NRAS lines than selu and bini. These results support investigating PAS-004 further and a clinical trial in patients with advanced solid tumors carrying RAS pathway mutations, including NRAS variants, is ongoing.

Digital droplet PCR-based detection of KRAS, NRAS, and BRAF mutation in plasma samples from patients with colorectal cancer.

e15626 Background: Digital droplet PCR (ddPCR) is known for high sensitivity to detect mutated DNA in plasma. The aim of this study was to investigate the concordance of KRAS, NRAS and BRAF mutation detection in patients with colorectal cancers (CRCs) between plasma samples (PSs) and tissue samples (TSs) in pair. Methods: We prospectively collected paired PS and TS from patients with CRC (N = 200) who underwent surgery or biopsy for metastasis at Seoul National University Boramae Medical Center from Mar 2017 to January 2020. The ddPCR was performed using QX200™ Droplet Digital PCR System (Bio-Rad, USA) according to the manufacturer's recommended protocol with PCR kits developed for each gene (Gencurix, South Korea). Results: A total of 187 TSs and PSs were valid for ddPCR. Among these, 111 had results for KRAS, 51 had results for NRAS and 21 had results for BRAF, which were tested through routine clinical practice (RCP) using the pyrosequencing or Sanger sequencing method. The experiment's outcomes were analyzed for KRAS, NRAS, and BRAF, respectively. The analysis initially compared the results of RCP TS and ddPCR TS, and subsequently, intended to compare the mutation detection rates between TS and PS by comparing the results of ddPCR TS and ddPCR PS for FFPE/PS paired specimens. For KRAS, compared to RCP TS, the sensitivity of ddPCR TS was 100%, specificity was 66.7%, and the overall concordance rate was 79.3%. In TS/PS paired specimens, the sensitivity of ddPCR PS for ddPCR TS results was 38.5%, specificity was 97.6%, and the overall concordance rate was 64.7%. For NRAS, compared to RCP TS, the sensitivity of ddPCR TS was 100%, specificity was 98.0%, and the overall concordance rate was 98.0%. In TS/PS paired specimens, the sensitivity of ddPCR PS for ddPCR TS results was 33.3%, specificity was 100%, and the overall concordance rate was 97.4%. For BRAF, sensitivity of ddPCR TS was not available (no RCP positive sample), specificity was 85.7% compared to RCP, corresponded to a concordance rate of 97.0%. The sensitivity of ddPCR PS for ddPCR TS results was 71.4%, specificity was 100%, and the overall concordance rate was 97.4%. The sensitivity of ddPCR PS for ddPCR TS results was 71.4%, specificity was 100%, and the overall concordance rate was 97.4%. Conclusions: The present study showed that ddPCR has high accuracy in detecting KRAS, NRAS and BRAF mutation in plasma samples from patients with CRC. Analysis of PS could be useful in early response assessment and longitudinal disease monitoring for patient with metastatic CRC with specific mutation.

Phase I study of GUCY2C CAR-T therapy IM96 in patients with metastatic colorectal cancer.

2518 Background: The clinical outcomes of metastatic colorectal cancer (mCRC) therapies are limited, especially for liver metastasis. Guanylyl cyclase 2C (GUCY2C) is ectopically expressed in all stages of CRC and intestinally restricted. GUCY2C-targeted CAR-T (IM96) was developed and phase I study was conducted to evaluate the safety and efficacy (NCT05287165). Methods: In this open-label, 3+3 dose-escalation study, IM96 was evaluated in GUCY2C-positive mCRC patients (pts) failed to ≥3 lines of therapies. Pts were pre-treated with fludarabine and cyclophosphamide, and received a single infusion of IM96 at the dose of 3×108 (DL1), 6×108 (DL2), 12×108 (DL3), or 20×108 (DL4) CAR-T cells. Dose-expansion study was performed at DL3. The primary objectives were safety and toxicity, and the secondary objectives were efficacy and pharmacokinetic profile. Results: As of December 2023, 20 pts were enrolled and infused with IM96. The follow-up time was 7-19 months for all pts. The median age was 52.5, and 11/20 cases were male. Liver metastasis was found in 11/20 cases (55%), proficient mismatch repair (pMMR) in 20/20 pts (100%), KRAS mutation in 12/20 pts (60.0%), NRAS mutation in 1/20 pts (5.0%), and BRAF mutation in 3/20 pts (15.0%). Bridging therapies were used in 19 pts. Only 1/20 pt (5.0%) showed neurotoxicity and ≥grade 3 cytokine release syndromes (CRS). Grade 1-2 CRS occurred in 16/20 pts (80.0%) with dramatic increase of interleukin-6. Grade 1-3 rash was observed in 14/20 pts (70.0%). Grade 3 diarrhea occurred in 11/20 pts (55.0%), and grade 1-3 oral mucositis appeared in 7/20 pts (35.0%), only in DL2, DL3 and DL4 groups. Dose-limiting toxicity and maximum tolerated dose were not achieved. Among 19 evaluable pts, the disease control rate (DCR) was 73.7%, and the objective response rate (ORR) was 26.3%. In DL3 group, pts showed an ORR of 40.0%, nevertheless of liver metastasis or not. The median progression-free survival time was 7 months, and the median duration of response was 10 months in DL3 group. No responding pts showed disease progression within 6 months. Tumor responses were correlated with significant decreases in carcinoembryonic antigen levels among all pts. Conclusions: This study demonstrated that IM96 has durable efficacy with acceptable safety profile in pMMR mCRC pts, in particularly, showing high therapeutic potential in liver metastasis pts. Clinical trial information: NCT05287165 .

FCN-159, a MEK1/2 inhibitor, in patients with advanced melanoma harboring NRAS or NF1mutations: A phase 1B dose-expansion study.

3095 Background: In phase 1A dose-escalation study, preliminary anti-tumor activity of FCN-159 was observed in advanced melanoma patients harboring NRAS mutations (ORR: 19.0%, mPFS: 3.8 months) with tolerable safety profile, which was posted during AACR 2022 (No.: 22-LB-7398-AACR). Here, we report the results from phase 1B dose-expansion study in advanced NRAS- or NF1-mutant melanoma patients at RP2D predetermined at 12 mg. Methods: This open-label, dose-expansion, phase 1B study (NCT03932253) consists of two cohorts, which enrolled patients with unresectable stage III/IV melanoma harboring NRAS or NF1 mutations, respectively. Patients received FCN-159 orally at 12 mg once daily (QD) continuously in 28-day cycles. The primary endpoint was anti-tumor activity. Results: As of July 17, 2023, 46 patients (32 patients with NRASmutations in Cohort 1 and 14 patients with NF1mutations in Cohort 2) were enrolled and received 12 mg FCN-159 QD. The median follow-up time was 8.6 months. Among 25 patients who failed in previous anti-PD-1 therapy in Cohort 1, 6 had confirmed partial responses (ORR, 24.0%; 95% confidence interval [CI], 9.4–45.1). Median DOR was 6.5 months (95% CI, 2.7–NE). Median progression-free survival (mPFS) was 3.6 months (95% CI, 1.8–5.5). Among 14 patients in Cohort 2, 1 had PR (ORR, 7.1%; 95% CI, 0.2-33.9) and mPFS was 2.8 months (95% CI, 1.8-5.5) (Table). Grade≥3 FCN-159-related treatment-emergent adverse events (TEAEs) were reported in 10 (21.7%) patients in both cohorts; the most common was blood creatine phosphokinase increased (13.0%). FCN-159–related serious AEs (SAEs) were reported in 3 (6.5%) patients, which were edema lower limb, cellulitis and pneumonia. Two (4.3%) patients discontinued treatment due to FCN-159 related TEAEs, including serous retinal detachment and herpes zoster. No FCN-159-related TEAEs led to death. Conclusions: FCN-159 showed encouraging efficacy data with well tolerated safety profile in patients with NRAS-mutant advanced melanoma, especially those who failed in previous anti-PD-1 therapy. Clinical trial information: NCT03932253 . [Table: see text]

Multicenter clinical trial of DNA markers for non-invasive differential diagnosis of benign and malignant melanocytic lesions.

e21550 Background: The differentiation between benign melanocytic neoplasms (nevi) and malignant melanocytic neoplasms (melanoma) presents a significant diagnostic challenge, necessitating the development of accurate and reliable diagnostic markers. This study aimed to evaluate the analytical characteristics of selected DNA markers for their efficacy in differentiating these conditions. Methods: A prospective analysis was conducted on sample groups obtained from participants of the multicenter clinical study, MoleMed. The study included 112 histological samples from 100 patients and a corresponding number of cytological samples, collected with the aim of evaluating DNA markers' sensitivity and specificity. DNA extraction followed a previously developed protocol, with concentration and quality assessed through microchip electrophoresis and real-time PCR. Molecular testing targeted mutations in "hotspots" of the BRAF and NRAS genes, as well as promoter mutations in the TERT gene, employing mutation-specific PCR amplification and Sanger sequencing for verification. Results: DNA extraction was successfully performed for 101 cytological samples, with concentrations ranging from &lt; 0.001 ng/µl to 5,508 ng/µl; the average concentration was 106.52 ng/µl, and the median was 2.37 ng/µl. BRAF mutations were found in 28 samples, with the mutation c.1799T &gt; A (p.Val600Glu, V600E) in 21 samples, c.1798_1799GT &gt; AA (p.Val600Lys, V600K) in 6 samples, and c.1799_1800TG &gt; AT (p.Val600Asp, V600D) in 1 sample. NRAS mutations were detected in 11 samples, including c.181C &gt; A (p.Gln61Lys, Q61K) in 6 samples, c.182A &gt; G (p.Gln61Arg, Q61R) in 3 samples, and c.182A &gt; T (p.Gln61Leu, Q61L) in 2 samples. No cases of simultaneous presence of more than one mutation in BRAF and NRAS "hotspots" within the same cytological sample were observed. DNA profiling of cytological material revealed signs of malignant neoplasms (MN) in 49 samples and no signs of MN in 52 samples. TERT mutations c.-124C &gt; T were detected in 21 samples, c.-146C &gt; T in 31 samples (including "borderline" cases), and c.-139_-138CC &gt; TT in 4 samples. Six samples exhibited more than one mutation, highlighting the complexity of genomic alterations in these neoplasms. Conclusions: The PCR analysis of the five DNA markers on both histological and cytological samples of human skin melanomas and nevi demonstrates promising analytical characteristics, with sensitivity and specificity exceeding 70% for all tested scenarios. These results highlight the potential utility of these DNA markers in the differential diagnosis of melanocytic neoplasms, offering a reliable, non-invasive diagnostic tool that complements traditional histopathological examination. Clinical trial information: NCT04353050 .

Dual-agent immunotherapy for prevention of melanoma brain metastases: A real-world analysis of 8686 patients.

2022 Background: Melanoma brain metastases (MBM) are a common endpoint among patients with advanced melanoma and prognosis is poor. Strategies for MBM prevention can prolong life and reduce morbidity. Dual agent immunotherapy (dIT) has been paradigm-changing in management malignant melanoma management. Recent clinical trials have supported the role of dIT for upfront management of small, asymptomatic MBM. Yet, its potential role in extending brain metastasis-free survival (BMFS) and decreasing MBM incidence overall has not been explored. The objective was to compare MBM incidence, median overall survival (OS), and BMFS in melanoma patients treated with dIT versus single immunotherapy (sIT). Methods: A real-world deidentified database collating clinical information from 92 organizations (TriNetX, Inc.) was queried. Melanoma patients without brain metastases prior to immunotherapy were stratified by treatment (anti-CTLA4 [sIT] and combination anti-CTLA4/ anti-PD1 [dIT]) and propensity-score matched. MBM incidence was measured within 5 years post-IT initiation. A complementary single-institution retrospective cohort study analyzed melanoma patients treated from 2012-2019. Median OS and BMFS were compared via log-rank tests and multivariate Cox proportional-hazards models. A competing risk analysis was performed to measure the cumulative incidences of MBM and death without MBM. Results: TriNetX identified 8,686 melanoma patients who received immunotherapy (4,585 dIT; 4,101 sIT). MBM incidence was 13.4%, and 19.3%, for the dIT and sIT cohorts, respectively (p &lt; 0.0001). DIT was associated with a lower likelihood of developing MBM compared to sIT (RR [95%CI], 0.69 [0.62-0.77]). After propensity-score matching on demographics and comorbidities, MBM incidence was similarly 13.3% and 18.5% for the dIT and sIT cohorts, respectively. On single-institution analysis, 130 melanoma patients were included (87 dIT; 43 sIT with anti-CTLA4). In patients with stage IV disease, median OS was 1.70 and 3.66 years for the sIT and dIT cohorts, respectively (p=0.6). Median BMFS was 1.56 and 2.36 years in the sIT and dIT cohorts respectively (p=0.76). On multivariable Cox proportional hazard regression analysis, NRAS mutation was significantly correlated with a worse prognosis for BMFS. Conclusions: These data highlight the impact of combination anti-CTL4/ anti-PD1 immunotherapy in decreasing MBM incidence. The potential primary prophylactic role of dIT in MBM warrants prospective exploration, including mechanistic understanding.

Characterization of driver oncogenic mutations of in-transit melanoma metastases.

9586 Background: In-transit melanoma (ITM) is a distinctive form of melanoma metastasis, marked by aggressive locoregional progression and characterized by the entrapment of tumor cells within the lymphatic channels before reaching regional lymph nodes (LN). The mechanisms leading to lymphatic trapping rather than progression to distant metastasis are not well-understood. Previous research indicates a clonal origin for ITM, suggesting specific early-stage genetic alterations may drive this clinical phenotype. In this study, we sought to identify driver oncogenic mutations specific to ITM that may serve as potential targets. Methods: We analyzed data from the MSK-IMPACT database, which includes targeted sequencing of 341-468 genes for patients with cutaneous melanoma. Patients were stratified into four groups based on the type of sample sequenced: in-transit, primary tumor, regional LN, and distant metastases. We examined the prevalence of driver mutations and genetic variants across these groups, adjusting for multiple comparisons. Results: A total of 528 patients were included, of which 74 were in the ITM, 103 in the primary, 133 in the regional LN, and 218 in the metastatic group. The mean age of the cohort was 59 years (SD = 16) and 67% were male. Among the in-transit samples, 68% were treatment-naïve at the time of sequencing, 36% received anti-PD1 monotherapy, and 12% received anti-PD-1 combination therapy. None of the patients received targeted therapy. NRAS Q61 driver mutations were significantly more common in ITM than all other groups (all p&lt;0.05). NF-1 driver mutations were significantly less common in ITM than distant metastasis (p=0.007) (Table). Comparative analysis across all genes showed a lower incidence of NF-1 mutations in ITM relative to metastatic samples (12% vs 38%, p=0.007). Gene interaction analysis showed mutual exclusivity between NRAS and PAK5 mutations in ITM (p=0.02), but not in other groups (p&gt;0.05). Conclusions: This study reveals distinct driver oncogenic mutations specific to ITM, characterized by a high incidence of NRAS mutations, retained NF-1 function and the novel finding of mutual exclusivity between NRAS and PAK5. Closer examination of the interplay between NRAS, NF-1 and PAK5 interactions in ITM may provide deeper insights into the molecular mechanisms of ITM and offer potential therapeutic targets for ITM. [Table: see text]

A phase III, open-label, multicenter, randomized controlled trial of tunlametinib versus investigator-selected chemotherapy in patients with advanced NRAS-mutant melanoma who had previously received immunotherapy.

TPS9606 Background: NRAS-mutant melanoma is an aggressive subtype with worse prognosis. However, no targeted therapy has been approved to date worldwide. Tunlametinib (HL-085) has showed an encouraging efficacy (confirmed ORR:34.8%, mPFS:4.2 months) with a manageable safety profile in phase II pivotal registrational study, which was published in ASCO 2023 annal meeting (NO.:9510). Here, we present the design of phase III randomized controlled study. Methods: This is a multicenter, two-arm, open-label, randomized controlled phase III confirmatory clinical trial to evaluate the efficacy and safety of tunlametinib in comparison with the combination chemotherapy of investigator's choice in advanced NRAS-mutant melanoma patients who had previously received immunotherapy. A total of 165 subjects from about 12 sites in China will be included and randomly assigned to the corresponding treatment group in 2:1 ratio. Two stratification factors before randomization: LDH level and whether have received chemotherapy. The key inclusion criteria included: a) Patients with unresectable stage III or metastatic IV melanoma confirmed by histology or cytology; b) History of immunotherapy failure or could not tolerate immunotherapy; c) Be able to provide NRAS mutation positive test report at baseline and provide sufficient histological specimens for NRAS mutation confirmation by central laboratory. Experimental group subjects will receive continuous administration of tunlametinib 12mg BID, 28 days per cycle. Control group subjects will receive one of the following regimens which according to investigator's choice (paclitaxel plus carboplatin, or temozolomide plus cisplatin, or dacarbazine plus cisplatin) , 28 days per cycle. The two groups will receive continuous therapy until disease progression or intolerable toxicity. The primary endpoint is progression-free survival which assessed by independent radiology review committee. Secondary endpoints included overall survival, objective response rate, disease control rate, duration of response, safety, pharmacokinetics and exposure-response. Exploratory endpoints are to evaluate quality of life between two groups and to development a companion diagnostic kit for NRAS gene mutations detection. This study is currently open for enrollment. Clinical trial information: NCT06008106 .

Characterization of necroptosis activators in colorectal cancer.

3522 Background: Necroptosis is a pro-inflammatory form of programmed cell death triggered by cellular stresses and extrinsic cytokines. It is a potential therapeutic target for immunotherapy (IO) in colorectal cancer (CRC). Here, we present comprehensive clinical and molecular characterization of key necroptosis regulators in CRC. Methods: 24,257 samples of CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq), NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES), and PD-L1 expression (22C3 clone; TPS ≥1%) were analyzed. Expression of activators in the necroptosis pathway ( RIPK3, MLKL, CYLD, and ZBP1) was categorized as either high (H) or low (L) using median expression as the cutoff. CRCs with high expression of all four activators were considered to have high propensity for necroptosis activity (NA-H, N=4595) compared to tumors with low expression in all activators (NA-L, N = 4946). RNA deconvolution analysis with QuantiSEQ estimated immune cell infiltration in the tumor microenvironment (TME). Differences in overall survival (OS) were analyzed from insurance claims data and calculated from treatment initiation using Kaplan-Meier estimates. Statistical significance was set as a P-value adjusted for multiple comparisons ( q&lt;0.05). Results: NA-H was associated with increased PD-L1 positivity (4.0% vs 2.3%), immune cell infiltration, and T-cell inflamed score (all q&lt;0.001). TP53 mutations were more frequent in NA-H, while SMAD2, SMAD4, KRAS, and NRAS mutations were associated with NA-L (all q&lt;0.01). NA-H was associated with activation of KRAS signaling, allograft rejection, inflammatory/interferon gamma response, and pancreatic beta cell function pathways (all q&lt;0.05). NA-H showed significantly improved OS in FOLFOX/FOLFIRI (F/F) (NA-L: 34.3 vs NA-H: 39.6 months [mo], P &lt; 0.001; HR = 0.83, 95% CI 0.77 – 0.90) and those treated with IO (15.3 vs 22.4 mo, P = 0.018; HR = 0.77, 95% CI 0.62– 0.96) treated CRC patients. The benefit remained in TP53-mutated CRC treated with F/F (34.6 vs 41.0 mo, P &lt; 0.001; HR = 0.82, 95% CI 0.74-0.90) and IO (10.0 vs 21.8 mo, P = 0.027; HR = 0.68, 95% CI 0.48-0.96) but not in the TP53-wild-type subgroup. Improved OS was observed with increased expression of CYLD and ZBP1(Table). The benefit remained in TP53-mutated CRC but not in the TP53 wildtype subgroup. Conclusions: This is the largest molecular and clinical characterization of necroptotic genes in CRC. Our data shows that activation of the necroptosis pathway is associated with immune cell infiltration/pathway activation, PD-L1 expression, and improved OS on F/F and IO. This benefit is more pronounced in TP53-mutated CRC, suggesting possible novel therapeutic targets.[Table: see text]

NRAS mutation in melanoma: Association of codon-specific changes on patient outcomes.

e21545 Background: NRAS mutant melanoma is an aggressive subtype making up 20-30% of cutaneous melanoma cases without specific treatment options beyond immune checkpoint inhibition (ICI). Previous studies have suggested specific NRAS codon mutations are associated with unique signaling pathways. To test the hypothesis that NRAS codon mutations in cutaneous melanoma may drive a preferential response to ICI, we evaluated specific genetic subsets of NRAS mutant melanoma based on the Q61 and G12/G13 codons. Methods: Cutaneous melanoma samples (n = 4091) were analyzed by NGS at Caris Life Sciences. Samples were stratified by NRAS status and subdivided into NRASQ61, NRASG12/13, and NRAS-wildtype. PD-L1 expression was assessed using IHC (positive ≥ 1%). TMB-high was defined as ≥ 10 mutations/Mb. Transcriptomic signatures of MAPK pathway activation and T-cell signatures were calculated. Real-world overall survival (rwOS) information was obtained from insurance claims data, with Kaplan-Meier estimates calculated from time on ICI until last date of contact. Mann-Whitney U, Chi-square, and Fisher exact tests were applied as appropriate, with p-values adjusted for multiple comparisons. Results: Cutaneous melanoma samples were comprised of 22% NRASQ61 (n = 902), 3.1% NRASG12/13 (n = 125) and 74.8% NRASWT (n = 3064), with the most common NRAS mutation being Q61R. Patients harboring NRASG12/13 codon changes were associated with concurrent mutations in BRAF (21.0 vs 6.4%, p&lt; 0.0001), NF1 (50.5% vs 8.7%, p&lt; 0.0001), KIT (3.2% vs 0.2%, p&lt; 0.001), ARID2 (22.1% vs 11.3%, p&lt; 0.01), MAP2K1 (7.2% vs 2.6%, p = 0.022) compared to NRASQ61. NRASG12/13 tumors were more frequently TMB-high compared to NRASQ61 or NRASWT (83.1% vs 62.1% vs 55.1%, p = 0.0001), but no difference was observed in the prevalence of dMMR/MSI-H or PD-L1 expression. Transcriptomic analysis showed that NRASQ61 mutants had a significantly higher MPAS compared to NRASG12/13 or NRASWT cohorts (Median: 1.16 vs 0.67 vs 0.46, p = 0.0147), while T-cell-inflamed scores were significantly higher in NRASWT compared to NRASQ61 (Median: 4 vs -40, p = 0.00492) but not to NRASG12/13 (Median: 4 vs -13.5, p = 0.674). No difference in rwOS was identified in NRASG12/13 and NRASQ61 melanoma treated with ICI (HR: 0.753, [95% CI: 0.43 – 1.31], p = 0.316). Conclusions: Patients with NRAS-mutated cutaneous melanoma exhibit codon-specific ( NRASQ61 vs NRASG12/13) signaling and prevalence of immunotherapy-related biomarkers. Our findings indicate that melanoma samples with NRASG12/13 mutation tend to exhibit immune richness suggesting they could be viewed as a distinct group that might potentially benefit from immune therapies. This observation offers valuable insight into codon-specific tumor subsets, differential response to therapy, resistance mechanisms, and the potential development of combination therapies to enhance survival outcomes of patients with NRAS mutant melanoma.

Results of a phase 2 study of evorpacept (E, ALX148), cetuximab (C), and pembrolizumab (P) in patients with refractory microsatellite stable metastatic colorectal cancer (MSS CRC).

3530 Background: Single-agent anti-PD-1/PD-L1 drugs are ineffective in MSS CRC. E is an engineered protein (high-affinity CD47-blocker fused to an inactive IgG Fc region), which blocks the CD47/SIRPα innate immune inhibitory phagocytosis checkpoint expressed on CRC and phagocytes, respectively. Preclinical evidence suggests ECP may be effective in MSS CRC. Methods: This phase 2, single-arm, two-stage, multicenter, investigator-initiated trial of E (15 or 10 mg/kg weekly at dose level [DL] 1 and -1, respectively), C (400 mg/m2 then 250 mg/m2 weekly), and P (200 mg every 3 weeks) in 21-day cycles enrolled patients with MSS CRC, regardless of tumor sidedness and RAS/BRAF status, refractory to ≥ 2 prior lines of therapy, including EGFR inhibitor if indicated (NCT05167409). All patients in both Stage 1 (safety run-in) and Stage 2 (expansion) received ECP until progression or unacceptable toxicity. The co-primary objectives were to determine the recommended dose (RD) of E with CP and objective response rate (ORR) by RECIST v1.1 (by one-sided exact test with α=0.05, H0 p ≤ 3% [historical controls], HA p ≥ 15%; power is 87% with N=48). Results: The safety-evaluable population included 16 patients, of whom 69% were male and median age 53 years. The primary tumor was in the right, left, and transverse colorectum in 31%, 31%, and 6% of patients, and was unknown in 31%. Mutations in KRAS, NRAS, and BRAF genes were present in 38%, 6%, and 0% of patients. The median number of prior lines of therapy was 3, including prior EGFR inhibitor in 44%. In Stage 1, 9 and 3 patients were enrolled at E DL1 and DL-1, respectively. E DL1 was established as the RD for Stage 2, in which 4 patients were enrolled. The most common any-grade treatment-emergent adverse events were headache (32%), fatigue (26%), anemia (21%), diarrhea (21%), dyspnea (16%), abdominal pain (16%), nausea/vomiting (16% each), and acneiform rash (16%). There were two on-study deaths assessed as related to all three study drugs (hemophagocytic lymphohistiocytosis and cytokine release syndrome; 1 each). While both are known associations with checkpoint inhibitors, other significant factors contributing to these event outcomes included cancer progression with high tumor burden in the former and patient decision to transition to comfort care in the latter. In the safety-evaluable population, ORR was 6.3% (1 ongoing partial response, 95% confidence interval [CI] 0.2-30.2%), disease control rate was 12.5% (95% CI 1.6-38.4%), median progression-free survival was 2.6 months (mo; 95% CI 2.1-2.9 mo), and median overall survival was 10.9 mo (95% CI 3.3 mo-infinity). Formal hypothesis testing was not performed. Conclusions: Further dose optimization of ECP is needed to establish a RD in the refractory MSS CRC population. While initial activity was seen, at the DLs evaluated, criteria for study termination were met. Clinical trial information: NCT05167409 .

The genetic risk architecture of mucosal melanoma.

10595 Background: Mucosal melanoma (MM) is a rare and aggressive subtype of melanoma. Outcomes for MM are significantly worse than for cutaneous melanoma (CM). While somatic mutations induced by UV radiation are known to cause CM, less is known about environmental and genetic alterations associated with MM. High penetrance alleles have been identified in familial cases of CM but the contribution of germline mutations to development of MM is not understood. Further, whilst population-based risk has been elaborated in CM, little is known for MM. The aim of this study is to characterize the germline genetic architecture of MM from high penetrance alleles to patient population genetics. Methods: A large retrospective cohort of MM patients (n=247) seen at MD Anderson Cancer Center were identified. Demographics, primary tumor sites, and presence of somatic mutations in BRAF, NRAS and KIT were extracted. Germline sequencing was performed on PBMC derived genomic DNA from the cohort using a gene panel of 322 major cancer genes. Frequency of identified germline alleles were compared to expected population control rate based on TOPMED using Fisher’s exact test. For genome wide association analysis (GWAS), genotyping will be performed on the MM cohort and the control group using the Infinium Global Screening Array. Cancer-free controls matched 3:1 based on MM cohort demographics have been assembled from existing case-control studies at MD Anderson. Following standard quality control procedures, we will impute variants from TOPMed using the Michigan Imputation Server. SNPtest v2.5.268 software will be utilized to perform association tests, using a multivariate logistic regression model incorporating gender and principal components. In addition to variants that may surpass a genome-wide significance score, we will inspect results for an enrichment of cancer genes. Results: Median age of MM cohort is 63, female predominant (60%), primarily White (84.6%), with gastrointestinal (36%), genitourinary (34%) and head &amp; neck (30%) being the most common MM subtypes. Characteristics of the cohort were representative of the US MM population. Somatic mutations in KIT (26%) were most common. Germline mutations were found in 10.9% of the cohort, with most common detected variations in MITF (c.G952A:p.E318K) (2.4%) at OR of 13.9 (95%CI 5.0, 30.9), and CHEK2 (c.1100delC:p.T367fs) (1.6%) at OR of 16.4 (4.4, 42.9) compared to population control rate. Conclusions: Our study is the first to demonstrate the distribution of germline mutations in the largest cohort of MM patients. The MITF E318K allele is a known risk factor for CM, while CHEK2 1100delC allele has mixed evidence for conferring CM risk. GWAS data are currently being analyzed and these updated results will be presented in the context of the comprehensive analyses of germline risk alleles in MM patients.

Comparing the efficacy and safety of HepaSphere drug-eluting bead transarterial chemoembolization combined with hepatic arterial infusion chemotherapy versus hepatic arterial infusion chemotherapy alone in unresectable colorectal liver metastases.

3565 Background: Drug-eluting bead transarterial chemoembolization (DEB-TACE) and hepatic arterial infusion chemotherapy (HAIC) are widely employed treatments for unresectable colorectal liver metastases (CRLM). Limited evaluation exists regarding TACE plus HAIC versus HAIC alone. This study aimed to evaluate the efficacy of combining HepaSphere DEB-TACE with HAIC compared to HAIC alone in patients with unresectable CRLM. Methods: A total of 259 CRLM patients aged over 18 years, treated at Peking University Cancer Hospital between March 2015 and November 2022, were included in this retrospective study. Among them, 157 patients received HepaSphere DEB-TACE combined with HAIC (DEB-TACE-HAIC), while 102 patients received HAIC alone. Propensity score matching (PSM) was performed to avoid selection bias and enable comparative analysis of efficacy and safety between the two cohorts. The primary endpoint was progression-free survival (PFS), while secondary endpoints comprised hepatic progression-free survival (hPFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR) and safety. Results: PSM resulted in 83 pairs, achieving comparable baseline between DEB-TACE-HAIC and HAIC cohorts. Of these pairs, 116 were male and 50 were female. Notably, 51.2% of patients harbored mutations in either KRAS, NRAS, or BRAF genes. Prior to enrollment, 94.6% of patients had received systemic treatments. Median hPFS, PFS and OS were all higher in the matched DEB-TACE-HAIC cohort (median hPFS: 7.6 vs. 4.1 months, P=0.023; median PFS: 5.1 vs. 3.5 months, P=0.092; median OS: 15.5 vs. 14.7 months, P=0.513). ORR was 37.3% in the DEB-TACE-HAIC group and 27.7% in the HAIC group, with DCR of 75.9% and 69.9%, respectively. There were no treatment related deaths. Serious adverse events were comparable between the two groups, except for nausea, which occurred more frequently in the HAIC group (39.8% vs. 22.9%, P=0.019). Conclusions: DEB-TACE-HAIC demonstrated superior survival compared to HAIC alone in unresectable CRLM, even in patients refractory to systemic therapy. The combination of HepaSphere DEB-TACE and HAIC appears promising as a regional treatment option with improved outcomes and a comparable safety profile.

Liquid biopsy multiomics enable clinical triage for early colorectal cancer diagnosis.

e15599 Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United State. Despite advancements in screening, diagnosis, and treatment, the disease remains a formidable public health issue. In 2023, approximately 153020 individuals were diagnosed with CRC and 52550 will die from the disease. In order to meet early diagnostic, we developed this multiomics assay to enable clinical triage for liquid biopsy screening. Methods: Xenonucleic acid (XNAs) are artificial genetic polymers that retain the Watson-Crick base-pairing capability and exhibit high chemical and biological stability. They can be employed as molecular clamps in RT-qPCR due to the stronger hybridizing/binding capability seen in XNA/DNA duplexes than DNA/DNA duplexes. We are developing an XNA-based multiplex RT-qPCR assay for APC, BRAF, CTNNB1, KRAS, NRAS, PIK3CA, SMAD4 and TP53 gene mutation panel and CRC 9 genes methylation panel combined with multiplex protein panel, called ColoscapeTM Plus. We enrolled in FIT positive subjects of both genders, aged 50-74, attending the CRC screening program, we tested 152 plasma samples, 75 health, 50 AA and 27 early CRC with this multiomics assay. Results: The assay results showed that its sensitivity was about 44% (95% CI: 0.302- 0.587) and specificity about 80% (95%CI: 0.689- 0.880) for advanced adenoma (AA). For CRC stage I and II, its sensitivity is about 63% and 78% with 80% specificity separately. Conclusions: The primary data has shown that this low cost and rapid turnaround time multiomics assay has a comparable sensitivity for early colorectal cancer diagnostic.

Neoadjuvant pembrolizumab and lenvatinib in resectable mucosal melanoma: NeoPlus study update.

9580 Background: Combination therapy of anti-PD1 agent with VEGFR inhibitor is a promising therapeutic approach in unresectable or metastatic mucosal melanoma. We conducted this study evaluating neoadjuvant lenvatinib and pembrolizumab in pts with resectable mucosal melanoma. Methods: This was a single-arm, open-label, single-center, phase 2 study conducted from Sep 2021. Eligible pts were adults (18-75 yr) with histologically confirmed, resectable mucosal melanoma. Pts received lenvatinib 20mg qd and pembrolizumab 200mg q3w for 2 cycles, followed by surgery. Pembrolizumab (200mg q3w) continued post operatively for further 15 cycles. The primary endpoint was complete pathologic response (pCR). Secondary endpoints were Relapse free survival (RFS, time from surgery to any event), Overall survival (OS) and safety. Results: Between Sep 2021 and Apr 2023, 26 pts were enrolled, median age was 57 yrs(range: 49-75), 19 were female. Primary sites included: 11 female genital, 8 ano-rectal, 5 head &amp; neck (1 nasal, 4 oral),2 esophageal. 15 pts (58%) were localized disease,11 (42%) were regional lymphatic disease. BRAF V600, NRAS or KIT mutations were presented in 1, 3 and 2 pts, respectively. 21 pts underwent surgery, 2 pts had a pCR (9.5%), 2 near PCR, 4 pPR, with a pathologic response rate of 38% (8/21,95%CI 18-62%). 5 pts did not proceed with planned surgery for pts preference. As of Dec 2023, 12 of 21 resected pts had relapsed (9/11 in female genital,2/4 in ano-rectal, 1/5 in head &amp; neck). Median follow up time was 19.1 months (95%CI 13.7-24.5). Median RFS was 12.4 months (95%CI 4.0-20.8), no difference was observed among these primary sites (female genital: 6.6 months, ano-rectal: 14.1 months, head &amp; neck: not reached). Median DMFS was not reached. IHC data of the resected tumor showed higher CD8+ T cells density in responders (R: pCR+near PCR+ pPR) than non-responders (NR: pNR) (p = 0.046). Tertiary lymphoid structures were detected in 9 pts. Most common TRAEs were hypertension (13, 50%), proteinuria (12, 46%), LDH increased (9,35%), hypothyroidism (8, 31%). Three pts experienced a dose reduction and 5 experienced a dose interruption due to an AE. No grade 4-5 toxicities were observed. Conclusions: Updated results confirmed that the combination of pembrolizumab plus lenvatinib as neoadjuvant therapy had moderate anti-tumor efficacy in resectable mucosal melanoma. Increased CD8+ T cell infiltration was observed in tumor with pathologic response, supporting further investigation in mucosal melanoma.

Atezolizumab (A), cobimetinib (C), and vemurafenib (V) in patients (pts) with BRAFV600 mutation–positive melanoma with central nervous system (CNS) metastases (mets): Final results and exploratory biomarker analysis from the phase 2 TRICOTEL study.

9550 Background: Primary analysis results from the phase 2 TRICOTEL study showed intracranial activity with the triplet combination of A + C + V in pts with BRAFV600-mutated melanoma with CNS mets (1). Here, we report final analysis results and exploratory biomarker analyses for pts with BRAFV600-mutated melanoma in the triplet combination cohort from TRICOTEL. Methods: Eligible pts were aged ≥18 y and had melanoma, MRI-confirmed CNS mets ≥5 mm in ≥1 dimension, and no prior systemic treatment for metastatic disease. Pts received A (840 mg on days 1 and 15 of each 28-d cycle) + C (60 mg once daily for 21 d on, 7 d off) + V (720 mg twice daily) except in cycle 1, during which A was withheld. Tumor tissue and circulating tumor DNA (ctDNA) samples collected at baseline (cycle 1, day 1) and ctDNA at the time of progressive disease (PD) were profiled for mutations using next-generation sequencing. Tumor tissue was profiled using a pipeline programmed for MelArray, and ctDNA was measured based on the tissue genetic profile. Results: A total of65 pts were enrolled in the triplet combination cohort. At final analysis, median follow-up was 12.4 mo. Per independent review committee (IRC) assessment, intracranial ORR (confirmed by assessments ≥4 wk apart) was 38% (95% CI, 27–51) and median intracranial PFS was 5.1 mo (95% CI, 3.7–6.9) (Table). Median OS was 13.4 mo (95% CI, 10.7–16.9). No new safety signals were observed since primary analysis. Mutations were detected in baseline ctDNA samples of 60 pts (5 pts not reported) and showed high prevalence of NF1, NRAS, and GNAI2 mutations; relative to tumor tissue, 11 additional BRAF mutations were detected in ctDNA at baseline. Acquired mutations in AKT1 were detected in ctDNA at PD. Median OS shortened in pts with &gt;2 versus ≤2 mutated MAPK pathway genes in ctDNA at baseline (9.1 vs 14.5 mo; hazard ratio, 2.0 [95% CI, 1.0–3.7]; p&lt;0.05). In tumor tissue, baseline mutations in PIK3C2A and PLEKHG4 were enriched in nonresponders, while RAD51B mutations were enriched in responders. Conclusions: Combination A + C + V had clinical intracranial activity in pts with BRAFV600-mutated melanoma with CNS mets. Exploratory biomarker analyses in this small cohort demonstrate the presence of ctDNA in pts with melanoma and CNS mets and provide insight into mutations associated with response and resistance to the triplet combination. 1. Dummer R et al, Lancet Oncol 2023. Clinical trial information: NCT03625141 . [Table: see text]

A phase 1 study of the BET inhibitor ZEN003694 in combination with the MEK1/2 inhibitor binimetinib in solid tumors with RAS pathway alterations and triple-negative breast cancer (NCI number 10449).

TPS3182 Background: Bromodomain and extra-terminal (BET) proteins serve as epigenetic readers and regulate transcription by binding acetylated lysines in histones. BET inhibitors target this epigenetic machinery modifying the expression of oncogenes and have the potential to overcome drug resistance in different settings. Resistance to mitogen-activated extracellular kinase (MEK) inhibition appears to rely upon changes in gene expression, a process that can be potentially inhibited via BET blockade. Preclinical data have demonstrated synergistic efficacy of dual MEK and BET inhibition in MAPK-altered solid tumors. Triple negative breast cancer (TNBC) is a disease frequently found to have gene amplifications in BRAF, NF1 mutations, and upstream regulators of the MAPK pathway and preclinical work has shown efficacy with dual inhibition of MEK and BET. Methods: This phase 1, open-label, multicenter study was designed to evaluate the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of the BET inhibitor ZEN003694 in combination with the MEK inhibitor binimetinib in solid tumors with RAS pathway alterations and TNBC. The study has two parts: dose escalation (part 1) which plans to recruit a maximum of 30 patients and dose expansion (part 2), which will comprise 12 patients. The primary objective of part 1 is to determine the MTD and recommended Phase 2 dose (RP2D), whereas the primary objective of dose expansion is to evaluate safety and toxicity. Secondary objectives include tolerability, pharmacokinetics (PK), and antitumor activity. Additional analyses will explore epigenetic changes such as ChIPseq to determine levels of H3K27ac and ATACseq to determine treatment-induced changes in open chromatin regions. ZEN003694 is given orally daily, and binimetinib is administered orally twice daily in 28-day cycles. Dose escalation is being conducted using a standard 3+3 cohort design to determine the MTD. Part 1 requires evaluable or measurable disease, whereas Part 2 requires measurable disease as per RECIST v1.1. Patients must have an ECOG performance status of ≤2. Included tumors are TNBC (estrogen receptor ≤1%, progesterone receptor ≤1%, human epidermal growth factor receptor 2 (HER2) 0–1+ or non-amplified) or any other solid tumor with actionable MAPK alterations (e.g. KRAS, NRAS, HRAS, or BRAF activating mutations, inactivating NF1 mutations, or BRAF fusions). Patients with any PI3K pathway activating genomic alterations are excluded. Prior therapy with BET, RAF, MEK, or ERK inhibitor is not permitted. This study is currently ongoing in dose escalation. Clinical trial information: NCI number 10449.