NRAS Q61 Mutations Research Articles

Association of various RAS codon mutations and prognostic outcomes of patients with colorectal liver metastases after hepatectomy.

The prognostic and predictive value of RAS mutations in patients with colorectal liver metastases (CRLM) who have undergone hepatectomy holds substantial importance. The present study aimed to investigate the impact of different RAS codon mutations on long-term survival in CRLM patients. A retrospective analysis was conducted on clinicopathological data from 399 CRLM patients with RAS mutations who underwent hepatectomy between January 2000 and December 2020. The RAS mutation gene status was assessed in KRAS codons (G12, G13, Q61, and A146) and NRAS codons (G12, G13, and Q61). Survival curves were generated using the Kaplan-Meier plotter and compared using the log-rank test. Univariate and multivariate analyses were performed to analyze the clinicopathological data. In the entire cohort, patients with KRAS G12 mutations exhibited the most favorable prognosis (p = 0.018). Comparatively, patients harboring KRAS Q61 mutations experienced poorer overall survival (OS) with a median of 15 months versus 33 months (p = 0.011) when compared to those with KRAS G12 mutations. Moreover, patients with NRAS Q61 mutations also showed decreased OS with a median of 26 months versus 33 months (p = 0.020) in comparison to KRAS G12 mutation patients. The results of multivariate analysis showed that both KRAS Q61 mutation (HR 2.130; 95% CI 1.088-4.168; p = 0.027) and NRAS Q61 mutation (HR 2.877; 95% CI 1.398-5.922; p = 0.004) were independent influencing factors of OS. Based on all identified risk factors, patients with RAS mutation were divided into high-risk and low-risk groups. Notably, in the high-risk group, the incorporation of postoperative chemotherapy was associated with longer OS, while it did not improve the survival of patients in the low-risk group. KRAS Q61 and NRAS Q61 mutations are promising predictors for OS in CRLM patients after hepatectomy. Postoperative chemotherapy may significantly benefit CRLM patients with RAS mutations, particularly those identified as high-risk.

Abstract 6942: Single cell resolution of intrapatient drug resistance mechanisms in metastatic melanoma

Abstract Intrapatient heterogeneity among different metastatic sites contributes to immunotherapy (ICi) and targeted therapy resistance. Determining the evolution of metastatic melanoma lesions that develop within the same patient could help elucidate adaptive mechanisms that drive tumor progression during therapy. To assess how metastatic melanoma persists through immunotherapy and targeted inhibitor treatments, we utilized samples from a single patient who had received anti-CTLA4, BRAFi/MEKi, and BRAFi/MEKi+CDK4/6i treatments during the LOGIC2 clinical trial. Eight short-term cultures were established from different metastatic site biopsies at chronological time points throughout the patient’s treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial treatment naïve metastatic tumor in the breast was BRAFV600E mutant, NRASQ61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. Tumors that persist through treatment and colonize distant organs adapt to and rely on organ-specific microenvironments. To assess treatment- and organ-specific adaptations, single-cell RNA sequencing was performed on seven of the short-term cultures to identify subpopulations that persist through each treatment. We analyzed samples and subpopulations of each culture to determine differences in their transcriptional cell states. The regulon transcriptional network of each sample and subpopulation was evaluated to determine site- and treatment-specific transcriptional regulation differences. In addition, we identified the treatment naïve culture is split into two major subpopulations by therapy resistance features which are further made up of six distinct subpopulations with heterogeneous regulatory networks. We determined the longitudinal evolution of subpopulations in response to treatment and organ site to evaluate how the pressure of therapies and the microenvironment influence metastatic progression. We further evaluated site-specific resistance mechanisms and detected increased HER3 expression in the melanoma brain metastasis (MBM) sample. Treatment with Neuregulin 1, a ligand for the HER3 receptor that is abundant in brain tissue, partially rescued the growth of MBM cells and restored MAPK and AKT-mTOR signaling in the presence of BRAFi/MEKi. Targeting the NRG1-HER3 signaling axis could have therapeutic potential in melanoma patients with active brain metastases. Additionally, we evaluated treatment-specific resistance mechanisms and identified high Brevican (BCAN) expression in an early emerging subpopulation that arises after ICi in this patient and in others. BCAN is implicated in both ICi- and cross-resistance. We will functionally investigate whether targeting BCAN sensitizes cells to ICi. Future studies aim to functionally examine novel site- and treatment-specific cross patient targets. Citation Format: Haley P. Wilson, Glenn L. Mersky, Jelan I. Haj, Jessica Teh, Phil F. Cheng, Signe Caksa, Casey D. Stefanski, Vivian Chua, Claudia Capparelli, Mitch P. Levesque, Reinhard Dummer, Timothy J. Purwin, Andrew E. Aplin. Single cell resolution of intrapatient drug resistance mechanisms in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6942.

Abstract A088: NST-628 is a potent, best-in-class MAPK pathway molecular glue that inhibits RAS- and RAF-driven cancers

Abstract Alterations in the RAS/RAF/MEK/ERK signaling cascade are common across multiple solid tumor types and aberrant signaling of the pathway is a driver for RAS- and RAF-driven cancers. Apart from approved mutation selective inhibitors for BRAF Class I and KRAS G12C mutations, other mutations of RAS and RAF are not directly addressable by currently approved inhibitors and there is a need for inhibitors with superior efficacy, durability and tolerability for the MAPK pathway in RAS- and RAF-driven cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents phosphorylation and activation of MEK by RAF, leading to deep durable inhibition of MEK kinase activity and downstream ERK signaling. In a MEK1 immunoprecipitation assay, NST-628 treatment glues ARAF, BRAF, and CRAF with unphosphorylated MEK1, stabilizing an inactive RAF-MEK complex and deeply inhibiting phospho-ERK signaling. In an unbiased cell line panel screen (550 models), NST-628 demonstrates efficacy across multiple tumor types with RAS-MAPK alterations, including melanoma, lung, and pancreatic models. NST-628 is broadly efficacious in models with BRAF Class II/III mutations, KRAS-mutations (G12C, G12D, G12V, G12R, Q61H), and NRAS-mutations (Q61x, G12x) as well as showing anti-proliferative effects in NF1-mutant/deficient models. Cell lines with NRAS Q61 mutations were particularly sensitive to NST-628 inhibition (GI50 average=150 nM). In vivo, NST-628 has a balanced metabolic profile and predicted half-life and exposure in humans is compatible with low dose daily dosing. NST-628 (3-5 mg/kg QD treatment) led to tumor regressions in HCT116 (KRAS G13D colorectal) and IPC-298 (NRAS Q61L melanoma) xenograft models, 53% and 38% respectively, correlating to inhibition of both phospho-MEK and phospho-ERK in tumors. In comparison to the MEK inhibitor trametinib, NST-628 led to deeper tumor responses and on target pathway inhibition in both models as well as significantly greater tolerability as measured by body weight. In a mini-mouse trial utilizing patient derive xenograft (PDX) tumors harboring NF1, KRAS G12D/R, BRAF Class II/III, and NRAS Q61x mutations, NST-628 (3 mg/kg QD) dosing demonstrates broad anti-tumor responses across melanoma, lung, pancreatic, glioma, and ovarian models. These data collectively, together with the predicted effective human half-life of NST-628 being consistent with daily dosing, support best-in-class potential for NST-628 and initiation of clinical trials in patients with solid tumors harboring RAS- and RAF-mutations is planned. Citation Format: Meagan B Ryan, Natasha Schenk, Marshall Zingg, Matthew Koehler, Brooke Swalm, Bradley Quade, Chaoyang Ye, Arvin C Dar, Yongxin Han, Klaus P Hoeflich, Michael R Hale, Margit Hagel. NST-628 is a potent, best-in-class MAPK pathway molecular glue that inhibits RAS- and RAF-driven cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A088.

Abstract 1301: Evolution of heterogenous BRAFi/MEKi+CDK4/6i resistance mechanisms and targeting site-specific resistant populations in metastatic cutaneous melanoma

Abstract Intrapatient heterogeneity among different metastatic sites contributes to targeted therapy resistance. Determining the proteomic profile of metastatic melanoma lesions that develop within the same patient could help elucidate adaptive mechanisms that drive tumor progression during therapy. Tumors that persist through treatment and colonize distant organs adapt to and rely on organ-specific microenvironments. To assess how protein expression is altered in cutaneous melanoma derived from different metastatic sites in a single patient, we utilized patient-derived BRAFi/MEKi and BRAFi/MEKi+CDK4/6i-treated samples from the LOGIC2 clinical trial. Eight cell lines were established from patient biopsies at chronological time points throughout treatment and subsequent post-mortem resection of the bone, ovary, and brain metastatic sites. While the initial metastatic tumor in the breast was BRAFV600E mutant, NRASQ61 mutations were acquired after treatment with CDK4/6i in the bone and ovary tumors. RPPA was performed on six of the cell lines to characterize site-specific differences in protein expression. We detected upregulation of ErbB3 in the brain metastatic tumor. Since neuregulin 1, a ligand for ErbB3, is abundant in brain tissue, we tested its ability to promote resistance to BRAFi/MEKi in melanoma brain metastases. Neuregulin 1 rescued the growth of brain metastasis cells and restored phospho-AKT and phospho-ERK1/2 levels in the presence of BRAFi/MEKi. Therefore, the ErbB3-Neuregulin axis may be targeted, for example by using ErbB3 inhibitors to improve response to MAPK inhibitors in melanoma brain metastases. Further studies aim to investigate the evolution of metastatic cutaneous melanoma longitudinally in response to targeted therapies. Site-specific signaling mechanisms will be functionally examined as possible avenues for metastasis-specific targeted therapies to improve patient outcomes. Citation Format: Haley P. Wilson, Timothy J. Purwin, Claudia Capparelli, Phil F. Cheng, Mitch P. Levesque, Reinhard Dummer, Jessica Teh, Andrew E. Aplin. Evolution of heterogenous BRAFi/MEKi+CDK4/6i resistance mechanisms and targeting site-specific resistant populations in metastatic cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1301.

RAS Mutations in Adult Acute Myeloid Leukemia (AML). Frequency, Mutational Spectrum, and Identification of a Comutation Bias for KRASK117 (TET2/ASXL1)

Introduction RAS mutations are subclonal events associated with both good and bad prognosis AML categories (Papaemmanuil E et al.N Engl J Med 2016; 374). An adverse prognostic meaning of RAS mutations in treated AML with myelodysplastic-related changes has also been reported.In solid tumors, it has been shown that patients with KRASQ61K do not confer resistance to therapy, unlike other mutations: KRASQ61K introduces a cryptic splice donor site and causes a short inactive isoform (Kobayashi Y et al. Nature 2022 ;603). However, most KRASQ61K patients acquired the silent mutation G60G, which removes the new splice donor site and increases therapy resistance. This splicing mechanism was not present in cases with NRASQ61K and HRASQ61K. We investigated the frequency of RAS mutations, their molecular associations, and the prevalence of splicing mechanisms involving KRASQ61K and NRASQ61K in AML. Methods We included a consecutive series of adult de novo AML enrolled in the CETLAM trials, a total of 620 patients who were diagnosed between 2017 to 2022. DNA and RNA were extracted from bone marrow or peripheral blood. Next Generation Sequencing (NGS) was carried out with three panels containing at least 42 AML-related genes, including KRAS and NRAS. Only variants with a VAF (variant allele frequency) >4% and ranked as pathogenic or likely pathogenic were used for the analysis. Conventional karyotype was routinely assessed. cDNA was obtained from 7 patients with NRASQ61K, and was amplified through a conventional PCR. cDNA products were observed in gel electrophoresis and sequenced by the Sanger method. IBM SPSS Statistics was used to perform Chi-Square tests. In statistical tests regarding molecular associations, patients with co-mutated NRAS and KRAS are excluded. Results Our study found RAS mutations in 16.9% (105 patients) of adult AML cases. 11.9% of AML patients had mutated NRAS, while 6.1% mutated KRAS (74 and 38 patients, respectively). In our study, Q61 was the third most common mutation in NRAS with an incidence of 26%, while it was the fourth most common in KRAS with a frequency of 12%. The distribution of mutations in KRAS and NRAS is shown in figure 1. Regarding the Q61 codon, the distribution of each amino acid change in our cohort is shown in Figure 1b. Nine NRASQ61K were found, and cDNA could be obtained from 7 patients. NRASQ61K and non-mutated NRAS samples presented the standard large transcript in the gel electrophoresis. These findings ruled out the presence of exon skipping in AML with NRASQ61K mutations. No single case of KRASQ61K was observed in this extensive series of AML, so we could not identify alternative transcripts described in solid tumors nor compensatory KRASG60G mutations.1.1% of patients (7 cases) presented co-mutated NRAS and KRAS. In 9 subjects, more than one NRAS mutation was found, whereas, in 3 patients we detected two KRAS mutations. Lastly, we focused on molecular associations of RAS mutated patients (Figure 2). Of the total RAS-mutated cohort (105 patients), DNMT3A and NPM1 had the highest mutation frequency. Notably, 40.3% of NRAS-mutated patients had NPM1 mutations, significantly higher (p=0.018) than KRAS (16.1%). Another interesting finding was that KRASK117N mutations were mutually exclusive with DNMT3A and NPM1. All KRASK117N (6 cases) were associated with TET2 and or ASXL1 mutations. Cytogenetics information was available for 87 patients with RAS mutations (59 NRAS and 35 KRAS). NRAS-mutated patients showed a higher frequency of core binding factor (CBF) alterations than KRAS (18.1% vs. 3.6%, respectively) (p=0.063). Complex and miscellaneous karyotypes had higher rates of KRAS mutations than NRAS mutations (57.1% vs. 32.7%, respectively, p=0.032).In 26 cases, RAS mutations were the single mutation or the one with the highest VAF; notably, 42.3% had an NRASQ61 mutation. We had cytogenetic information of 20 patients, and twelve had inv(16) (60%). Conclusions In this cohort of adult AML cases, RAS mutations were present in 16.9%. Mutations at codon G12 were the most prevalent lesions in AML. NRAS mutations were more frequent than those of KRAS. Up to 17% of RAS mutated AML cases showed more than one mutation. CBF chromosomal abnormalities were commonly associated with NRAS mutations. Complex and miscellaneous karyotypes showed KRAS mutations more often. KRASK117N mutations were consistently associated with TET2 and ASXL1 mutations and mutually exclusive with NPM1 and DNMT3A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ODP465 BRAF V600 and NRAS Q61 mutations in thyroid fine-needle aspiration (FNA) with indeterminate cytology in Argentina

Abstract It is known that thyroid cancer initiation and progression occurs because of gradual accumulation of genetic and epigenetic alterations at cell level. Many mutations associated with this disease have been reported to be present in genes encoding proteins in the mitogen-activated protein kinase (MAPK) signaling pathway, specifically BRAF and RAS genes, as well as in the PI3K/Akt signaling pathway, among others. In particular, molecular alterations in follicular cells in the BRAF or NRAS genes have been reported to be associated with the process of carcinogenesis. Previously we found BRAF and NRAS mutations are observed in a significant number of papillary and follicular thyroid carcinomas in our population, respectively. In this study, we tested the diagnostic performance of BRAF and NRAS mutation in thyroid carcinoma in fine-needle aspiration (FNA) specimens with indeterminate cytology. In total, residual material from 73 FNA samples was used (n= 16, Bethesda III; n=48, Bethesda IV and n=9 Bethesda V). BRAF codon 600 mutation and NRAS codon 61 mutations were investigated using qPCR and melting curve analysis was carried out. Diagnosis of malignancy was confirmed by histology on paired surgical specimen in 54 cases. Results In the cytologically indeterminate categories, 9.6% of specimens were BRAF V600 mutated: 1 of 16 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (6.3%); 2 of 48 as follicular neoplasm or suspicious for follicular neoplasm (4.1%); and 4 of 9 as suspicious for malignancy (44.4%). NRAS Q61 mutation was found in 4 patients (2 with Bethesda IV and 2 with cytology V). The BRAF V600 mutations were associated with carcinoma in 100% of cases (n = 7), whereas only 75% (n = 3) of the nodules with NRAS were associated with carcinoma. BRAF V600 and NRAS Q61 mutations had high specificity in predicting malignant carcinoma (96.8%); the sensitivity was 43.5% with a positive predictive value of 90.9% and a negative predictive value of 69.8%. In specific categories of indeterminate cytology, the pre-test risk of malignancy was 38%, 33% and 88% while the post molecular test risk of malignancy was 100%, 75% and 100% respectively. Conclusions BRAF V600 and NRAS Q61 molecular test in residual FNA samples improve diagnostic accuracy of the cytology analysis in our population and may help to better select patients for avoid inadequate or unnecessary surgeries. Presentation: No date and time listed

Prevalence and detection of actionable BRAF V600 and NRAS Q61 mutations in malignant peripheral nerve sheath tumor by droplet digital PCR

Malignant peripheral nerve sheath tumors ( MPNSTs) are aggressive tumors with poor prognosis that do not typically respond well to standard chemotherapy. Recently, point mutations involving BRAF V600E have been demonstrated in a subset of MPNST, offering the possibility of targeted treatment. However, the reported prevalence of these alterations is variable. Mutations involving NRAS, which is also involved in the MAPK/ERK pathway and amenable to targeted inhibitors, have not been well characterized in MPNST. In this study, we validated droplet digital polymerase chain reactionfor the detection of BRAF V600E and NRAS Q61 mutations and evaluate the prevalence of BRAF V600E and NRAS Q61 mutations in 79 cases of MPNST, including 45 sporadic, 27 NF-1 associated, and 7 radiation-associated tumors. We detected actionable BRAF or NRAS mutations in 3 of 44sporadic MPNSTs (6.8%), including 2 BRAFV600 and 1 NRAS Q61 mutations, as well as 1 NRAS Q61 mutation in a tumor that was ultimately considered to represent melanoma. These 3 cases with positive mutations were exclusively in sporadic, high-grade MPNST (FNCLCC grade 3 of 3), with a prevalence of 11.5% in this group (3.8% NRAS Q61 mutations and 7.7% BRAF V600 mutations). None of the tumors associated with NF-1 or prior radiation had detectable mutations in the genes tested. Overall, the prevalence of these alterations offers the possibility of targeted therapy in this aggressive type of sarcoma and suggests the potential benefit of routine clinical testing.

Associations between TERT Promoter Mutations and Survival in Superficial Spreading and Nodular Melanomas in a Large Prospective Patient Cohort

Survival outcomes in melanoma and their association with mutations in the telomerase reverse transcriptase gene TERT promoter remain uncertain. In addition, few studies have examined whether these associations are affected by a nearby common germline polymorphism or vary on the basis of melanoma histopathological subtype. We analyzed 408 primary tumors from a prospective melanoma cohort for somatic TERT-124[C>T] and TERT-146[C>T] mutations, the germline polymorphism rs2853669, and BRAFV600 and NRASQ61 mutations. We tested the associations between these variants and clinicopathologic factors and survival outcomes. TERT-124[C>T] wasassociated with thicker tumors, ulceration, mitoses (>0/mm2), nodular histotype, and CNS involvement. In a multivariable model controlling for the American Joint Committee on Cancer stage, TERT-124[C>T] was an independent predictor of shorter recurrence-free survival (hazard ratio= 2.58, P= 0.001) and overall survival (hazard ratio= 2.47, P= 0.029). Patients with the germline variant and TERT-124[C>T]-mutant melanomas had significantly shorter recurrence-free survival than those lacking either or both sequence variants (P < 0.04). The impact of the germline variant appeared to be more pronounced in superficial spreading than in nodular melanoma. No associations were found between survival and TERT-146[C>T], BRAF, or NRAS mutations. These findings strongly suggest that TERT-124[C>T] mutation is a biomarker of aggressive primary melanomas, an effect that may be modulated by rs2853669.

Prevalence and Detection of Actionable BRAF V600 and NRAS Q61 Mutations in Malignant Peripheral Nerve Sheath Tumor by Droplet Digital PCR

Prevalence and Detection of Actionable BRAF V600 and NRAS Q61 Mutations in Malignant Peripheral Nerve Sheath Tumor by Droplet Digital PCR

BRAFV600E Mutant Allele Frequency (MAF) Influences Melanoma Clinicopathologic Characteristics.

Simple SummaryThe mutational load of BRAFV600E in melanomas has been described as a possible prognostic biomarker but there is no information about the mutant allele frequency (MAF) variability of BRAFV600E within cutaneous melanomas and its potential prognostic implications. Our study suggests that the variation degree of BRAFV600E MAF within primary cutaneous melanoma could act as a determinant in the location of primary melanomas as well as their first metastases and could influence prognostic indicators such as Breslow and mitotic indexes. BRAFV600E MAF variation is also related to the neoplastic cell phenotype and tumor lymphocytic infiltrate of primary tumors. For all these reasons, detection of BRAFV600E MAF variation could be a useful prognostic biomarker. It is worth exploring the role of BRAFV600E MAF variation with regards to the response of melanoma to targeted therapies.Background: Cutaneous melanoma shows high variability regarding clinicopathological presentation, evolution and prognosis. Methods: Next generation sequencing was performed to analyze hotspot mutations in different areas of primary melanomas (MMp) and their paired metastases. Clinicopathological features were evaluated depending on the degree of variation of the BRAFV600E mutant allele frequency (MAF) in MMp. Results: In our cohort of 14 superficial spreading, 10 nodular melanomas and 52 metastases, 17/24 (71%) melanomas had a BRAFV600E mutation and 5/24 (21%) had a NRASQ61 mutation. We observed a high variation of BRAFV600E MAF (H-BRAFV600E) in 7/17 (41%) MMp. The H-BRAFV600E MMp were all located on the trunk, had lower Breslow and mitotic indexes and predominantly, a first nodal metastasis. Regions with spindled tumor cells (Spin) and high lymphocytic infiltrate (HInf) were more frequent in the H-BRAFV600E patients (4/7; 57%), whereas regions with epithelial tumor cells (Epit) and low lymphocytic infiltrate (LInf) were predominant (6/10; 60%) and exclusive in the low BRAFV600E MAF variation tumors (L-BRAFV600E). The H-BRAFV600E/Spin/HInf MMp patients had better prognostic features and nodal first metastasis. Conclusions: The H-BRAFV600E MMp were located on the trunk, had better prognostic characteristics, such as lower Breslow and mitotic indexes as well as high lymphocytic infiltrate.

A prospective, comprehensive registry that integrates the molecular analysis of pediatric and adolescent melanocytic lesions.

Childhood melanocytic tumors represent a diagnostic and therapeutic challenge, and additional research is needed to better define the natural history of these tumors. The authors developed a comprehensive, prospective registry called Molecular Analysis of Childhood Melanocytic Tumors for children and adolescents with an atypical Spitz tumor/Spitz melanoma (AST/SM), conventional or adult-type melanoma (CM), melanoma arising in a giant congenital nevus (MCM), or atypical melanocytic proliferation of other types (OT) to better define the clinical behavior of these lesions by incorporating an integrated clinicopathologic and molecular analysis using centralized pathology review and various platforms, including fluorescence in situ hybridization; array comparative genomic hybridization; and whole genome, exome, and capture targeted panels. From May 2016 to November 2019, 70 children were enrolled with a median age at diagnosis of 9.1 years. Thirty-seven had AST/SM, 17 had CM, 4 had MCM, and 12 had OT. Patients with AST/SM were younger (median age, 7 years), and their tumor most commonly arose in the extremities and trunk. The most common gene rearrangements included MAP3K8 and ALK. None of the 33 patients who underwent a TERT promoter mutation analysis had a mutation, and all patients were alive. Among the CM patients, the median age was 13 years; 11 had a BRAFV600E mutation, and 7 had a TERT promoter mutation. Three patients died of their disease. All 4 patients with MCM harbored an NRASQ61 mutation and died of their disease. The OT group was heterogenous, and all patients survived. The incorporation of an integrated clinicopathologic and genomic analysis identifies distinct subgroups of pediatric melanocytic lesions that have different clinical behaviors. The integration of this combined diagnostic modality can help to individualize diagnoses and treatments for these patients.

Abstract 2272: Interrogating RAS isoform and mutational specificity for effectors with bioluminescence resonance energy transfer (BRET)

Abstract Mutations in the small GTPases HRAS, NRAS, and splice variants KRAS4a and KRAS4b occur in roughly 20% of all cancers1. Each of these four RAS isoforms share a high degree of homology, yet play distinct biological roles; mouse models devoid of KRAS die mid-gestation, whereas HRAS and NRAS null mice survive2. The occurrence of RAS isoform mutations and their specific oncogenic codon substitutions are non-uniform across cancer types, with KRAS mutations in the G12 position frequently driving colorectal cancer while NRAS Q61 mutations are commonly present in skin cancer1. Several explanations have been proposed for this, including differential regulation at the level of transcription, mRNA stability, translation, and protein stability, along with distinctions in post-translational modification, localization, and tissue-specific availability of effectors3. Subtle differences in the biochemical properties of amino acid substitutions may also influence the dominant mutational forms across cancer types. However, the question of whether RAS isoforms and their specific codon substitutions preferentially engage with certain effectors within a cellular context remains unclear, despite considerable efforts. Here, we utilize bioluminescence resonance energy transfer (BRET) between mVenus- and NanoLuc-tagged recombinant proteins to derive quantitative measurements of effector affinity across RAS isoforms and common mutants. By transiently expressing a fixed concentration of NanoLuc-tagged effectors while titrating increasing amounts of mVenus-tagged RAS in a cellular system, we generated saturation curves that reflect the nature of binding between the protein pair in vivo to help untangle this longstanding question. We further validated interactions by immunoprecipitation, and determined construct expression and colocalization via western blot and confocal microscopy. 1Prior IA, Hood FE, Hartley JL. The frequency of Ras mutations in cancer. Cancer Research 20202Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nature Reviews Cancer 2003;3:459-653Li S, Balmain A, Counter CM. A model for RAS mutation patterns in cancers: finding the sweet spot. Nature Reviews Cancer 2018;18:767-77 Citation Format: Megan Rigby, John Columbus, Vanessa Wall, Dominic Esposito, Thomas Turbyville. Interrogating RAS isoform and mutational specificity for effectors with bioluminescence resonance energy transfer (BRET) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2272.

Abstract A46: A pan-cancer RAS mutant library elucidates the transformation potential of RAS variants

Abstract RAS mutations are highly prevalent in tumors, especially those of the lung, colon, skin and pancreas. The tissues in which these tumors arise are enriched for specific RAS isoform and codon preferences. For example, mutations in KRAS are prevalent in lung, colon and pancreas, while NRAS mutations are predominantly found in melanoma. Additionally, mutations in KRAS predominantly occur at the hotspot codons G12 or G13 and NRAS mutations predominantly occur at the hotspot codon Q61. Independent of the RAS isoform, the mutated codon and specifically the amino acid substitution at that codon contribute to distinct biochemical differences. We aim to address if different amino acid substitutions at hotspot codons induce differential transformation, i.e., KRAS G12C versus KRAS G12D. Further, we aim to address if different codons are disposed to differential transformation capabilities and if these are isoform specific, for example, if KRAS Q61 mutations are similar to KRAS G12 mutations and if KRAS Q61 mutations are similar to NRAS Q61 mutations. Additionally, we aim to characterize the transforming capacity of RAS alterations outside of hotspot codons. Hundreds of sporadic insertions and point mutations in RAS have been observed clinically, yet most of these alterations have not been experimentally investigated. To address these questions, we created a pooled library of 184 RAS (KRAS, NRAS and HRAS) alterations identified in 29,000 tumors from a panel of ~100,000 pan-cancer tumors. These alterations include missense, insertion and deletion mutations irrespective of the incidence in which they occur. The library contains the greatest representation of KRAS alterations, 104. Using this pooled library, we measured the relative proliferation effect of these RAS variants using Ba/F3 cells in a competition assay. In interleukin 3 (IL-3)-dependent murine pro B-cells, Ba/F3, expression of well-characterized RAS hotspot mutations led to IL-3-independent proliferation. Due to the robust Ba/F3 cellular transformation we observed upon expression of RAS variants, we sought to use this assay for the pooled-library screen. The Ba/F3 system enables us to interrogate the transformation capacity of the RAS alterations and to identify the relative strength of these mutations while assaying independent of tissue type. Citation Format: Amanda R. Moore, Jean-Philippe Fortin, Michael Costa, Marinella Callow, Benjamin Haley, Gerard Manning, Ethan S. Sokol, Shiva Malek. A pan-cancer RAS mutant library elucidates the transformation potential of RAS variants [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A46.

STAT3 Relays a Differential Response to Melanoma-Associated NRAS Mutations.

Melanoma patients carrying an oncogenic NRAS mutation represent 20% of all cases and present worse survival, relapse rate and therapy response than patients with wild type NRAS or with BRAF mutations. Nevertheless, no efficient targeted therapy has emerged so far for this group of patients in comparison with the classical combination of BRAF and MEK inhibitors for the patient group carrying a BRAF mutation. NRAS key downstream actors should therefore be identified for drug targeting, possibly in combination with MEK inhibitors. Here, we investigated the influence of different melanoma-associated NRAS mutations (codon 12, 13 or 61) on several parameters such as oncogene-induced senescence, cell proliferation, migration or colony formation in immortalized melanocytes and in melanoma cell lines. We identified AXL/STAT3 axis as a main regulator of NRASQ61–induced oncogene-induced senescence (OIS) and observed that NRASQ61 mutations are not only more tumorigenic than NRASG12/13 mutations but also associated to STAT3 activation. In conclusion, these data bring new evidence of the potential tumorigenic role of STAT3 in NRAS-mutant melanomas and will help improving current therapy strategies for this particular patient group.

RAS/RAF mutations in tumor samples and cell-free DNA from plasma and bone marrow aspirates in multiple myeloma patients.

Purpose: To evaluate the detection of gene mutations in bone marrow biopsy and circulating free DNA (cfDNA) from plasma in multiple myeloma (MM).Experimental design: We used cell-free DNA from plasma and bone marrow to test BRAF V600, KRAS G12/G13, NRAS G12/G13 and NRAS Q61 mutations using multiplex assays for droplet digital PCR (ddPCR), and evaluated results with clinical outcomes.Results: We found of 83 patients, the detectable mutation frequencies for the above four genes were 4 (5%), 13 (16%), 3 (4%) and 14 (17%) in bone marrow, respectively. The median variant allelic frequency (VAF) in most mutations were 1.595%. In 17 paired cfDNA samples, the detectable mutation frequencies for the above four genes were 5 (30%), 1 (6%), 0 (0%) and 3 (18%) respectively, and the median VAF rate was 2.9%. Agreement between bone marrow DNA and plasma cfDNA were 76%, 100%, 100% and 100% compared to the tissue detections, respectively. In 17 patients with paired bone marrow and plasma samples, the above four mutations were 3 (18%), 1 (6%), 0 (0%) and 2 (12%) respectively, with the agreement rates of 88%, 88%, 100% and 100% compared to tissue detections. Of 57 patients with available outcome data, high mutation VAF had a shorter median survival than patients with low mutation VAF (P=0.0322).Conclusions: Oncogenic mutations in BRAF, KRAS and NRAS genes can be detected in the bone marrow and plasma cfDNA with ddPCR in patients with MM patients and high VAF is associated with short survival.

Genetic landscape of adult Langerhans cell histiocytosis with lung involvement

The clinical significance of the BRAF V600E mutation in adult Langerhans cell histiocytosis (LCH), including pulmonary Langerhans cell histiocytosis (PLCH), is not well understood. Similarly, the spectrum of molecular alterations involved in adult LCH has not been fully delineated. To address these issues, we genotyped a large number of adult LCH biopsies and searched for an association of identified molecular alterations with clinical presentation and disease outcome.Biopsies from 117 adult LCH patients, 83 with PLCH (median age 36.4 years, 56 females, 38 multisystem disease, 79 single system disease, 65 current smokers) were genotyped for the BRAF V600E mutation. In 69 cases, LCH lesions were also genotyped by whole-exome sequencing (WES) or targeted gene panel next-generation sequencing (NGS). Cox models were used to estimate the association of baseline characteristics with the hazard of LCH progression.MAPK pathway alterations were detected in 59 out of 69 cases (86%) (BRAF V600E mutation: 36%, BRAF N486_P490 deletion: 28%, MAP2K1 mutations: 15%, isolated NRAS Q61 mutations: 4%), while KRAS mutations were virtually absent in PLCH lesions. The BRAF V600E mutation was not associated with LCH presentation at diagnosis, including smoking status and lung function, in PLCH patients. BRAF V600E status did not influence the risk of LCH progression over time.Thus, MAPK alterations are present in most lesions from adult LCH patients, particularly in PLCH. Unlike reports in paediatric LCH, BRAF V600E genotyping did not provide additional information on disease outcome. The search for alterations involved in the MAPK pathway, including BRAF deletions, is useful for guiding targeted treatment in selected patients with refractory progressive LCH.

Integrated phosphoproteomics and transcriptional classifiers reveal hidden RAS signaling dynamics in multiple myeloma

AbstractA major driver of multiple myeloma (MM) is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employed an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in MM to inform precision medicine strategies. Unbiased phosphoproteomics initially revealed differential activation of kinases across MM cell lines and that sensitivity to mammalian target of rapamycin (mTOR) inhibition may be particularly dependent on mTOR kinase baseline activity. We further noted differential activity of immediate downstream effectors of Ras as a function of cell line genotype. We extended these observations to patient transcriptome data in the Multiple Myeloma Research Foundation CoMMpass study. A machine-learning–based classifier identified surprisingly divergent transcriptional outputs between NRAS- and KRAS-mutated tumors. Genetic dependency and gene expression analysis revealed mutated Ras as a selective vulnerability, but not other MAPK pathway genes. Transcriptional analysis further suggested that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs wild-type RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes, whereas RAS mutations overall carry no survival impact. We further developed an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. These results will lead to improved stratification of MM patients in precision medicine trials while also revealing unexplored modes of Ras biology in MM.

1339P - Integrative RNAseq and target panel sequencing reveals common and distinct innate and adaptive resistance mechanisms to BRAF inhibitors

BackgroundTargeted therapy in melanoma has been a great success in extending progression free survival and overall survival for melanoma patients. These include BRAF inhibitors for patients with a BRAF V600 mutation and MEK inhibitors for patients with an NRAS G12 or Q61 mutation. However, only about 50 % of patients respond to single BRAF inhibitor therapy and about 70% respond to combination BRAF and MEK inhibitor therapy. MethodsWe in vitro tested each cell line for resistance to BRAF inhibitor and found 27 to be resistant. To elucidate the possible resistance mechanisms, we performed RNAseq and targeted panel sequencing on all 53 cultures and found specific subgroups of gene expression and mutations that define the innate and adaptive resistance populations. ResultsSurprisingly, a few melanoma cultures from patients who have never been exposed to BRAF inhibitors had innate resistance, while the majority of cell cultures from progressive patients were resistant to in vitro BRAF inhibition. . One surprising finding was that the phenotype switching signature was one of the resistant mechanisms in common for innate and adaptive resistance, suggesting a selection process for resistant cells during therapy. We also found mechanisms of resistance specific for adaptive resistance, there were many samples that gain mutations in NRAS or acquired a splicing event in BRAF that was not seen in the untreated tumor suggesting a de novo mechanism to resistance. ConclusionsOverall, we noticed several mechanisms to innate and adaptive resistance which highlights tumor and patient heterogeneity when treated with BRAF inhibitors and reinforces the concept for precision medicine in the treatment of melanoma. Legal entity responsible for the studyThe authors. FundingEU Horizon 2020 PHC grant No. 633974 (SOUND – Statistical multi-Omics UNDerstanding of Patient Samples). DisclosureR. Dummer: Honoraria (self): Novartis; Honoraria (self): Merck Sharp & Dhome; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Roche; Honoraria (self): Amgen; Honoraria (self): Takeda; Honoraria (self): Pierre Fabre; Honoraria (self): Sun Pharma; Honoraria (self): Sanofi. All other authors have declared no conflicts of interest.

CtDNA as a noninvasive monitoring tool in metastatic melanoma.

9548 Background: The field of liquid biopsy provides a promising alternative to standard tissue biopsies. Previous work has shown that plasma circulating cell-free DNA (ctDNA) can reflect the heterogeneous spectrum of mutations in cancer including metastatic melanoma. Our project aimed to establish and statistically validate plasma-based assays for tumour load and therapy monitoring in melanoma. Methods: On a large cohort of stage III and stage IV melanoma patients (N = 96) who received signalling targeted or immune checkpoint inhibitors we showed that the most common oncogenic drivers of this disease such as the BRAFV600E, NRASQ61 and the TERTC250T and TERTC228T promoter mutations (termed TERTprom) can be analysed in ctDNA with highly sensitive droplet digital PCR technology (detection of mutant ctDNA down to 0.01% analytical sensitivity). Results: Our research has demonstrated that ctDNA (irrespective of the genotype) significantly correlates with tumour stage (P &lt; 0.05). Using receiver operating characteristics (ROC) analyses thresholds were established for risk stratification and response prediction. Elevated ctDNA at baseline was a significant predictor of disease progression compared to elevated LDH or S100 in multivariable cox proportional hazards model (Hazard ratio [HR] 7.43, P = 0.05). During therapy, patients with low ctDNA load (below the ROC threshold) had significantly better radiological outcomes and prolonged progression free survival (PFS) compared to patients with high ctDNA load (P &lt; 0.0001). Our findings were confirmed on an independent cohort of metastatic melanoma patients (N = 35) treated with immune checkpoint inhibitors, where also during therapy low ctDNA load correlated with prolonged PFS (P = 0.003). An added benefit of ctDNA was demonstrated in about 80% of the patients, where ctDNA analyses preceded the radiological diagnosis of response or relapse. Progression was detected in plasma ctDNA in average 3.5 months earlier as compared to routine imaging techniques. Finally, we demonstrated that the occurrence of NRASQ61 mutation in BRAFV600-inhibitor treated patients at therapy baseline was associated with treatment failure. The sub-clonal NRASQ61 mutation at therapy baseline was an independent predictor of shorter PFS (HR 2.69, P = 0.02) as compared to BRAFV600E patients without the NRASQ61 mutation at therapy baseline. Conclusions: In sum, our results support the value of ctDNA as a sensitive biomarker for real-time therapy monitoring and early detection of disease progression.

High incidence of brain metastases (BrM) in patients with metastatic cutaneous melanoma (MCM) and mutations in the APC/β-catenin (CTNNB1).

9527 Background: Activation of the WNT/β-catenin pathway is associated with low/absent tumor-infiltrating lymphocytes (TILs) and resistance to anti-PD-L1/anti-CTLA-4 agents in mouse melanoma models. We aimed to investigate if APC/CTNNB1 mutations in melanoma pts are associated with TIL status, prediction of response to immunotherapy (IT), and overall survival (OS). Methods: Pts with CM and APC or CTNNB1 mutations identified in melanoma tumors using the TruSight Tumor 26 Illumina assay were enrolled. Demographics, clinical (stage, treatment response, follow-up), pathologic (TIL status), and molecular (BRAF/NRAS, C→T (i.e. UV signature) nucleotide transition, functional significance by IMPACT, mutant allele frequency (MAF) corrected from the % tumor) characteristics were investigated. Results: We identified a total of 25 pts (13 males; age at original diagnosis (median 61 yrs, range 22-78 yrs). CTNNB1 and APC mutations were mutually exclusive. 48% (12/25) had APC mutations and 52% (13/25) had CTNNB1 mutations; of which (i.e. CTNNB1 mutations) 69% (9/13) had absent TILs. 88% (22/25) of APC/CTNNB1 mutations had moderate functional significance, 64% (16/25) of the mutations had a C→T nucleotide change, 36% (9/25) had BRAFV600, and 20% (5/25) NRASQ61 mutations. 64% (14/22) of pts with stage II-III progressed to stage IV; of these 14 pts, 8 (57%) developed parenchymal BrM. 13 of the stage II-III 22 pts who progressed to stage IV received IT; of these 13 pts, 7 (53%) had absent TILs. APC/CTNNB1 mutations did not influence response to IT, irrespective of the MAF of the mutations. Of the 12 pts with MCM and measurable disease who received IT 9/12 had absent TILs and 7/12 responded. The median OS from the time of diagnosis of distant MCM (N = 17; 14 pts who progressed from initial stage II-III and 3 pts who were originally diagnosed with stage IV) was 18.8 months (range, 2.4-48.0 months). Conclusions: APC &amp; CTNNB1 mutations are mutually exclusive. CTNNB1 mutations are more frequently associated with absent TILs. Pts with MCM had relatively shorter OS (18.8 months) in part due to development of BrM. In this small cohort APC/CTNNB1 mutations did not seem to impair response to immunotherapy.