NRAS Mutations Research Articles

Genomic Alterations and Clinical Characterization in Chinese Patients with Metastatic Colorectal Cancer.

Metastatic colorectal cancer (mCRC) is increasingly characterized by myriad genomic alterations beyond the well-known factors such as RAS, BRAF, and microsatellite instability (MSI). Novel genomic changes, including ERBB2 amplifications, mutations, and gene fusions, are now recognized as potential targets for precision therapy. This study aims to explore the genomic landscape of a Chinese cohort with mCRC to identify potentially targetable genetic alterations for personalized treatment strategies. A total of 500 mCRC patients in China were enrolled, based on which genomic profiling was performed using capture-based targeted sequencing across a panel of 520 genes on tumor tissues to identify prevalent genomic alterations. The mutations were analyzed by optimized proprietary algorithms. MSI and mismatch repair deficiency status were analyzed using the read-count-distribution approach. Besides, the overall survival (OS) related to these molecular changes was estimated. The cohort's genomic profiling revealed TP53 mutations in 78%, APC in 60%, and KRAS in 47% of the patients. MSI-High status was confirmed in 5.8% of cases via a next-generation sequencing (NGS)-based algorithm. ERBB2/HER2 amplifications were found in 12% (60/500) of patients, with potential therapeutic implications for those without concurrent KRAS mutations. A subset of patients (1.2%; 6/500) showed fusions and DNA damage response (DDR) gene mutations (except TP53) that could be targeted therapeutically. The KRAS (G12C) variant was detected in 14 patients (2.8%), and 61 (12.2%) had a BRAF V600E mutation. Notably, survival analysis showed no significant differences in OS between KRAS mutant loci and NRAS mutations (p = 0.436). However, BRAF V600E mutations were associated with a poorer prognosis than BRAF wild-type and non-V600E mutations (16.3 months vs. 29.5 and 31.1 months, respectively; p < 0.001). This study validates the feasibility of using NGS to detect prognostic and therapeutically actionable genetic variants in Chinese mCRC patients, contributing to understanding the genomic variation within this population and highlighting the potential for personalized medicine in managing mCRC.

Clinical, Imaging, and Pathological-Molecular Characteristics Associated with Stage IA Invasive Lung Adenocarcinoma Recurrence After Sub-lobar Resection

Rationale and ObjectivesThis study aimed to investigate the association of clinical, imaging, and pathological-molecular characteristics with the prediction of patient prognosis with stage IA invasive lung adenocarcinoma (ILADC) after sub-lobar resection. Materials and MethodsThis study assessed 360 patients, including 91 and 269 with and without recurrence 3 years postoperativel, respectively, with stage IA ILADC undergoing preoperative chest computed tomography (CT) scans and subsequent sub-lobar resection at our institution. Their clinical and CT features and histological subtypes and gene mutation status were compared. Binary logistic regression analysis was conducted to identify the independent risk factors for recurrence. An external validation cohort included 113 patients, used to test the model’s efficiency. ResultsFor clinical features, old age, male gender, smokers, and high age-adjusted Charlson comorbidity index (ACCI) were frequently observed in patients with recurrence than those without (all p < 0.05). For CT features, large tumor size, solid-predominant density, spiculation, peripheral fibrosis, type II pleural tag, and pleural adhesion were more common in recurrent patients than non-recurrent ones (all p < 0.05). The regression model revealed old age, large tumor size, solid-predominant density, spiculation, type II pleural tag, and pleural adhesion as independent risk factors for recurrence, with an area under the curve (AUC) of 0.942. The external validation cohort obtained an AUC of 0.958. For phological-molecular features, miropapillary/solid-predominant growth pattern, KRAS, ALK, and NRAS mutation or fusion were more ccommon in the recurrent group, whereas EGFR mutation was more frequent in the non-recurrent group (all p < 0.05). ConclusionClinical and CT features help predict the prognosis of patients with stage IA ILADC after sub-lobar resection and decide for individualized treatment. Moreover, patients with different prognosis demonstrated different pathological-molecular features.

Мутации в генах KRAS/NRAS и эффективность индукционной терапии по трехкомпонентным схемам на основе бортезомиба у пациентов с впервые диагностированной множественной миеломой

AIM. To identify the KRAS and NRAS gene mutations in patients with newly diagnosed multiple myeloma (ММ) and to classify them according to the depth of antitumor response to bortezomib-based triplet induction therapy. MATERIALS &amp; METHODS. The trial enrolled 89 patients with newly diagnosed MM prior to chemotherapy. Among them, there were 45 women and 44 men aged 30–82 years (median 58.5 years). ММ was diagnosed according to IMWG criteria (2014). Bone marrow (BM) plasma cells were isolated from the aspirate using gradient method with subsequent immunomagnetic CD138 marker selection. The KRAS and NRAS gene mutations in BM CD138+ cells were identified with Sanger sequencing method. The proteomic programs MutationTaster, Polyphen2, and FATHMM-XF were used for mutation analysis in the KRAS and NRAS genes. All patients received bortezomib-based triplet chemotherapy as first-line treatment. The response depth was assessed after completing 6 cycles of PAD and VCD regimens. Antitumor response was evaluated according to IMWG (2016) criteria. RESULTS. The mutation rate in the gene family RAS was 42 % (37/89). The analysis focused on the data from 33 patients with mutations detected and response identified after 6 cycles of treatment. In 22 out of 33 patients, deep response was not achieved, whereas 11 patients showed complete remission (CR) + very good partial remission (VGPR). In the group of patients without mutations in the gene family RAS, the response to therapy meeting the CR + VGPR criteria was 64 % (27/42). The differences appeared to be significant (p = 0.008). The clinical data and the evaluation of primary treatment outcomes provided the basis for distinguishing a group of 9 prognostically unfavorable mutations: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val. CONCLUSION. The mutations in KRAS and NRAS belonging to the gene family RAS had a negative effect on the efficacy of the bortezomib-based triplet induction therapy. Mutation variants in the RAS family genes differed in prognostic significance. The analysis results helped to identify the mutation variants associated with the worse response to therapy: NRAS Gly13Asp, Gln61His; KRAS Gly12Ala, Gly12Asp, Gly12Val, Gly13Asp, Gln61Arg, Gln61His, and Ala146Val.

A novel colorectal cancer test combining microsatellite instability and BRAF/RAS analysis: Clinical validation and impact on Lynch syndrome screening

BackgroundLynch syndrome (LS) is under-diagnosed. UK National Institute for Health and Care Excellence guidelines recommend multistep molecular testing of all colorectal cancers (CRCs) to screen for LS. However, the complexity of the pathway has resulted in limited improvement in diagnosis.MethodsOne-step multiplex PCR was used to generate sequencing-ready amplicons from 14 microsatellite instability (MSI) markers and 22 BRAF, KRAS, and NRAS mutation hotspots. MSI and BRAF/RAS variants were detected using amplicon-sequencing and automated analysis. The assay was clinically validated and deployed into service in northern England, followed by regional and local audits to assess its impact.ResultsMSI analysis achieved 99.1% sensitivity and 99.2% specificity and was reproducible (r = 0.995). Mutation hotspot analysis had 100% sensitivity, 99.9% specificity, and was reproducible (r = 0.998). Assay-use in service in 2022–2023 increased CRC testing (97.2% (2466/2536) versus 28.6% (601/2104)), halved turnaround times, and identified more CRC patients at-risk of LS (5.5% (139/2536) versus 2.9% (61/2104)) compared to 2019–2020 when a multi-test pathway was used.ConclusionA novel amplicon-sequencing assay of CRCs, including all biomarkers for LS screening and anti-EGFR therapy, achieved >95% testing rate. Adoption of this low cost, scalable, and fully automatable test will complement on-going, national initiatives to improve LS screening.

The effect of genetic alterations detected by the circulating tumor DNA-based next-generation sequencing technique on prognosis and survival in metastatic colorectal cancer

Purpose: Studies conducted to date showed that circulating tumor DNA (ctDNA)-based next generation sequencing (NGS) panels are beneficial in the treatment strategies of patients with metastatic colorectal cancer (mCRC). In this study; we planned to determine the frequencies of various genetic alterations in patients with mCRC by ctDNA-based NGS analyses, evaluate the concordance rates by comparing these results with the results in standard polymerase chain reaction (PCR) analyses, and investigate the effect of the detected alterations on overall survival and progression-free survival. Materials and methods: The study was conducted by retrospective screening and analysis of the data on 48 patients, who were followed up with a diagnosis of mCRC and who received chemotherapy and/or biological agents. The data were analyzed using SPSS 25.0 [IBM SPSS Statistics 25 software (Armonk, NY: IBM Corp.)] package program. Results: In this study, ctDNA-based NGS analyses, compared to the quantitative PCR-based gold standard method, were found to have a sensitivity rate of 64.7%, specificity rate of 55.6% and concordance rate of 59.1% for KRAS mutation; a sensitivity rate of 100%, specificity rate of 86.7% and concordance rate of 87.1% for NRAS mutation; a sensitivity rate of 50%, specificity rate of 96.4% and concordance rate of 90.6% for BRAF mutation. In addition, concordance rates were evaluated based on to the time elapsed between the time of taking the liquid biopsy and tissue biopsy samples. As a result, concordance rates for KRAS, NRAS, and BRAF mutations were found to be 60.9%, 100%, and 100% respectively, in cases where this elapsed time was less than 6 months; and were found to be 57.1%, 78.9%, and 85% respectively, in cases where this elapsed time was more than 6 months. Furthermore, the comprehensive analyzes revealed that the frequency of many molecular changes in mCRC as well as the relationship of these changes with clinicopathological features and survival times. Conclusion: Our study demonstrates the clinical benefit of ctDNA-based NGS analyzes in patients with mCRC.

Predictors of response to immunotherapy in colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer-related deaths globally. While treatment advancements have improved survival rates, primarily through targeted therapies based on KRAS, NRAS, and BRAF mutations, personalized treatment strategies for CRC remain limited. Immunotherapy, mainly immune checkpoint blockade, has shown efficacy in various cancers but is effective in only a small subset of patients with CRC with deficient mismatch repair (dMMR) proteins or high microsatellite instability (MSI). Recent research has challenged the notion that CRC is immunologically inert, revealing subsets with high immunogenicity and diverse lymphocytic infiltration. Identifying precise biomarkers beyond dMMR and MSI is crucial to expanding immunotherapy benefits. Hence, exploration has extended to various biomarker sources, such as the tumor microenvironment, genomic markers, and gut microbiota. Recent studies have introduced a novel classification system, consensus molecular subtypes, that aids in identifying patients with CRC with an immunogenic profile. These findings underscore the necessity of moving beyond single biomarkers and toward a comprehensive understanding of the immunological landscape in CRC, facilitating the development of more effective, personalized therapies.

Adjuvant treatment with anti-PD-1 in acral melanoma: A nationwide study.

Previous studies demonstrated limited efficacy of immune checkpoint inhibitors in unresectable acral melanoma (AM); it remains unclear how this translates to the adjuvant setting. This study investigates clinical outcomes of acral compared to cutaneous melanoma (CM) patients treated with adjuvant anti-PD-1 after complete resection. All stages III-IV AM and CM patients receiving adjuvant anti-PD-1 after complete resection between 2018 and 2022 were included from the prospective nationwide Dutch Melanoma Treatment Registry. We analyzed recurrence-free survival (RFS), distant metastasis-free survival (DMFS), and overall survival (OS). A multivariable Cox regression analysis of RFS was performed to adjust for potential confounders. We included 1958 (86 AM and 1872 CM) patients. At baseline, AM patients more frequently had KIT mutations, higher disease stages, and Eastern Cooperative Oncology Group Performance Status, and fewer BRAF and NRAS mutations. Median RFS was 14.8 months (95% confidence interval [CI]: 11.5-29.3) in AM and 37.4 months (95% CI: 34.6 to not reached) in CM (p = .002). After correcting for potential confounders, AM remained associated with a higher risk of recurrence (HRadj 1.53; 95% CI: 1.07-2.17; p = .019). Two-year DMFS tended to be worse for AM than for CM: 64.5% versus 79.7% (p = .050). Two-year OS was significantly lower in AM (71.5% vs. 84.3%; p = .027). The results of this study suggest a poorer outcome of adjuvant-treated AM compared to CM. Studies assessing the added value of adjuvant treatment in AM are needed. Future research should investigate alternative treatment strategies to improve outcomes of high-risk AM.

Risk factors for synchronous high-risk polyps in patients with colorectal cancer.

Colorectal cancer (CRC) patients may experience inadequate preoperative colonoscopy due to bowel obstruction or inadequate bowel preparation, leading to potential oversight of other polyps. We aimed to identify risk factors for CRC complicated with synchronous high-risk polyps. A retrospective analysis of 6,674 CRC patients from December 2014 to September 2018 was conducted. High-risk polyps were defined as adenomas or serrated polyps that were ≥10 mm, or with tubulovillous/villous components or high-grade dysplasia. All other polyps were defined as low-risk polyps. Patients with complete pathological and clinical information were categorized into three groups: the no polyp group, the low-risk polyp group, and the high-risk polyp group. Univariate and multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and corresponding 95% confidence intervals (CIs) for all potential risk factors. Among the 4,659 eligible patients, 848 (18.2%) were found to have low-risk polyps, while 675 (14.5%) were diagnosed with high-risk polyps. In a multivariate logistic regression model, compared to patients without polyps, those with synchronous high-risk polyps were more likely to be male (OR = 2.07), aged 50 or older (OR = 2.77), have early-stage tumors (OR = 1.46), colon tumors (OR = 1.53), NRAS mutant tumors (OR = 1.66), and BRAF wild-type tumors (OR = 2.43). Our study has identified several risk factors associated with the presence of synchronous high-risk polyps in CRC patients. Based on these findings, we recommend that patients who exhibit these high-risk factors undergo early follow-up of colonoscopy to detect synchronous polyps early.

IMMU-04. THE ROLE OF CHECK POINT INHIBITORS IN PEDIATRIC PRIMARY DIFFUSE LEPTOMENINGEAL MELANOMATOSIS: REPORT OF TWO CASES

Abstract BACKGROUND Primary diffuse leptomeningeal melanomatosis (PDLM), a subtype of malignant melanomas of CNS, is rare, especially in children. PDLM is defined by diffuse invasiveness of the leptomeninges with or without nodules. Clinical manifestations of PDLM are nonspecific, mimicking the diagnosis of meningitis. As PDLM is neither sensitive to chemotherapy nor radiotherapy, the prognosis is extremely poor. Recently, some new therapeutic aspects like immunotherapy, especially immune check point inhibitors (ICI) are emerging. Cases report: First case: A 13 years old boy presented with 4 months of weakness, dysphasia and vomiting. MRI demonstrated diffuse leptomeningeal enhancement of the brain and spine. Brain biopsy confirmed the diagnosis of PDLM, with low PDL1 stains in immunohistochemistry, without targetable alterations on NGS. PET FDG normal. Treatment with ICI (Nivolumab 1 mg/kg/dose and Ipilimumab 3 mg/kg/dose, 4 courses every 21 days) was started. He developed severe complications after the 1st course (ICI enterocolitis, pancreatitis) and progressive disease after 2 courses. Craniospinal radiotherapy was delivered at a dose of 30 Gy. The disease continued to progress; the patient died nine months after diagnosis. Second case: A 7-year-old boy presented with diplopia, headache and vomiting. MRI demonstrated a cranial leptomeningeal enhancement and hypo-intense T1 and hyper-intense T2 signal at bilateral medial frontal cortex area. PET FDG normal. Brain biopsy confirmed the diagnosis of PDLM, with TMB 1.9, NRAS mutation and wild type BRAF by NGS. He started combination of ICI therapy. Three weeks after 1st course of Ipilimumab-Nivolumab he was admitted to PICU with prolonged status epilepticus. MRI demonstrated progression of leptomeningeal disease. Trametinib was deferred by parents and the child succumbed a year from diagnosis. CONCLUSIONS We report two cases of pediatric patients with PDLM treated unsuccessfully by immune check point inhibitors, emphasizing the diagnostic and therapeutic challenges in such a rare disease.

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 .