NRAS Mutations Research Articles

Phase I/II Study of Combined BCL-xL and MEK Inhibition with Navitoclax and Trametinib in KRAS or NRAS Mutant Advanced Solid Tumors.

MEK inhibitors (MEKi) lack monotherapy efficacy in most RAS-mutant cancers. BCL-xL is an anti-apoptotic protein identified by a synthetic lethal shRNA screen as a key suppressor of apoptotic response to MEKi. We conducted a dose escalation study (NCT02079740) of the BCL-xL inhibitor navitoclax and MEKi trametinib in patients with RAS-mutant tumors with expansion cohorts for: pancreatic, gynecologic (GYN), non-small cell lung cancer (NSCLC), and other cancers harboring KRAS/NRAS mutations. Paired pretreatment and day 15 tumor biopsies and serial cell-free (cf)DNA were analyzed. A total of 91 patients initiated treatment, with 38 in dose escalation. Fifty-eight percent had ≥3 prior therapies. A total of 15 patients (17%) had colorectal cancer, 19 (11%) pancreatic, 15 (17%) NSCLC, and 32 (35%) GYN cancers. The recommended phase II dose (RP2D) was established as trametinib 2 mg daily days 1 to 14 and navitoclax 250 mg daily days 1 to 28 of each cycle. Most common adverse events included diarrhea, thrombocytopenia, increased AST/ALT, and acneiform rash. At RP2D, 8 of 49 (16%) evaluable patients achieved partial response (PR). Disease-specific differences in efficacy were noted. In patients with GYN at the RP2D, 7 of 21 (33%) achieved a PR and median duration of response 8.2 months. No PRs occurred in patients with colorectal cancer, NSCLC, or pancreatic cancer. MAPK pathway inhibition was observed in on-treatment tumor biopsies. Reductions in KRAS/NRAS mutation levels in cfDNA correlated with clinical benefit. Navitoclax in combination with trametinib was tolerable. Durable clinical responses were observed in patients with RAS-mutant GYN cancers, warranting further evaluation in this population.

Abstract A107: Characterizing the genomic and immunologic landscape of serous borderline tumors and low-grade serous ovarian cancer

Abstract A107: Characterizing the genomic and immunologic landscape of serous borderline tumors and low-grade serous ovarian cancer

CtDNA improves prognostic prediction for patients with relapsed/refractory MM receiving ixazomib, lenalidomide, and dexamethasone

AbstractIt remains elusive how driver mutations, including those detected in circulating tumor DNA (ctDNA), affect prognosis in relapsed/refractory multiple myeloma (RRMM). Here, we performed targeted-capture sequencing using bone marrow plasma cells (BMPCs) and ctDNA of 261 RRMM cases uniformly treated with ixazomib, lenalidomide, and dexamethasone in a multicenter, prospective, observational study. We detected 24 and 47 recurrently mutated genes in BMPC and ctDNA, respectively. In addition to clonal hematopoiesis–associated mutations, varying proportion of driver mutations, particularly TP53 mutations (59.2% of mutated cases), were present in only ctDNA, suggesting their subclonal origin. In univariable analyses, ctDNA mutations of KRAS, TP53, DIS3, BRAF, NRAS, and ATM were associated with worse progression-free survival (PFS). BMPC mutations of TP53 and KRAS were associated with inferior PFS, whereas KRAS mutations were prognostically relevant only when detected in both BMPC and ctDNA. A total number of ctDNA mutations in the 6 relevant genes was a strong prognostic predictor (2-year PFS rates: 57.3%, 22.7%, and 0% for 0, 1, and ≥2 mutations, respectively) and independent of clinical factors and plasma DNA concentration. Using the number of ctDNA mutations, plasma DNA concentration, and clinical factors, we developed a prognostic index, classifying patients into 3 categories with 2-year PFS rates of 57.9%, 28.6%, and 0%. Serial analysis of ctDNA mutations in 94 cases revealed that TP53 and KRAS mutations frequently emerge after therapy. Thus, we clarify the genetic characteristics and clonal architecture of ctDNA mutations and demonstrate their superiority over BMPC mutations for prognostic prediction in RRMM. This study is a part of the C16042 study, which is registered at www.clinicaltrials.gov as #NCT03433001.

Restrospective reappraisal of the prognostic classification of spitzoid melanocytic neoplasms after BRAF and NRAS mutation characterisation: a single institution experience.

The current WHO classification of melanocytic tumours excludes neoplasms showing BRAF or NRAS mutations from the Spitz category. This study aimed to review and reclassify atypical melanocytic tumours with spitzoid morphological features diagnosed between 2009 and 2021 in our hospital after expanding the molecular profile, including BRAF and NRAS mutations in all cases. A total of 71 neoplasms showing spitzoid features (Spitz-like) and atypia were included. The risk of progression of tumours was first studied by integrating the morphology, immunohistochemistry (p16, Ki67, HMB45 and PRAME) and fluorescence in-situ hybridisation (FISH) results (melanoma multiprobe and 9p21). In a second step, after expanding the molecular study, including BRAF and NRAS mutational status, the neoplasms were finally classified into four subgroups: atypical Spitz tumour (AST, n = 45); BRAF-mutated naevus/low-grade melanocytoma with spitzoid morphology (BAMS, n = 2); Spitz melanoma (SM, n = 14); and BRAF or NRAS mutated melanoma with spitzoid features (MSF, n = 10). Follow-up of patients revealed uneventful results for AST and BAMS. Only one SM presented lymph node metastasis after 134 months. Conversely, patients with MSF showed an unfavourable outcome: three developed lymph node metastases after a mean time of 22 months, with one patient presenting distant metastasis and dying of the disease 64 months from diagnosis. The progression-free survival showed significant differences between the four groups of spitzoid tumours (P < 0.001) and between both melanoma subtypes (P = 0.012). The classification and prognostication of atypical neoplasms with spitzoid features requires the integration of histomorphology with the molecular investigation of tumours, which should include BRAF and NRAS mutational status.

Tumor location matters, next generation sequencing mutation profiling of left-sided, rectal, and right-sided colorectal tumors in 552 patients

Despite the introduction of new molecular classifications, advanced colorectal cancer (CRC) is treated with chemotherapy supplemented with anti-EGFR and anti-VEGF targeted therapy. In this study, 552 CRC cases with different primary tumor locations (250 left side, 190 rectum, and 112 right side) were retrospectively analyzed by next generation sequencing for mutations in 50 genes. The most frequently mutated genes were TP53 in left-sided tumors compared to right-sided tumors and BRAF in right-sided tumors compared to left-sided tumors. Mutations in KRAS, NRAS, and BRAF were not detected in 45% of patients with left-sided tumors and in 28.6% of patients with right-sided tumors. Liver metastases were more common in patients with left-sided tumors. Tumors on the right side were larger at diagnosis and had a higher grade (G3) than tumors on the left. Rectal tumors exhibit distinctive biological characteristics when compared to left-sided tumors, including a higher absence rate of KRAS, NRAS, and BRAF mutations (47.4% in rectal versus 42.8% in left-sided tumors). These rectal tumors are also unique in their primary metastasis site, which is predominantly the lungs, and they have varying mutation rates, particularly in genes such as BRAF, FBXW7, and TP53, that distinguish them from tumors found in other locations. Primary tumor location has implications for the potential treatment of CRC with anti-EGFR therapy.

Genetic landscape of 482 thyroid carcinomas: analysis with the national datacenter for cancer genomic medicine in Japan.

Comprehensive genomic profiling is useful for patients with Thyroid carcinoma (TC) for whom standard treatment has become refractory. We analyzed the clinical and genomic characteristics of patients with TC using the Japanese nationwide Center for Cancer Genomics and Advanced Therapeutics (C-CAT) database. This retrospective observational study used the data obtained from the C-CAT database. Genomic information has been accumulated on representative gene mutations associated with TC. Among the 482 patients, 212 (44%) were male and 270 (56%) were female. According to histological type, 259 (54%), 46 (10%), 16 (3%), 51 (11%), and 110 (23%) patients had papillary TC (PTC), follicular TC, medullary TC, poorly differentiated TC, and anaplastic TC (ATC), respectively. Among the genomic profiling tests, FoundationOne CDx (n = 388; 80%) was the most frequently performed. The frequencies of BRAF, NRAS, HRAS, KRAS, and RET mutations were 259 (54%), 62 (13%), 13 (3%), 16 (3%), and 12 (2%), respectively. The BRAF V600E mutation (n = 257) was the predominant BRAF mutation. TERT promoter mutations, which are associated with tumor aggressiveness, were detected in 308 patients (64%). PTC was the most common histologic type of TC for which genetic profiling was performed in Japan, followed by ATC. Since the most common targetable mutation is the BRAF mutation, practical application of BRAF-targeted therapy can be an important treatment option for Japanese patients with TC.

Fluoropyrimidine type, patient age, tumour sidedness and mutation status as determinants of benefit in patients with metastatic colorectal cancer treated with EGFR monoclonal antibodies: individual patient data pooled analysis of randomised trials from the ARCAD database

BackgroundKRAS mutations in metastatic colorectal cancer (mCRC) are used as predictive biomarkers to select therapy with EGFR monoclonal antibodies (mAbs). Other factors may be significant determinants of benefit.MethodsIndividual patient data from randomised trials with a head-to-head comparison between EGFR mAb versus no EGFR mAb (chemotherapy alone or best supportive care) in mCRC, across all lines of therapy, were pooled. Overall survival (OS) and progression-free survival (PFS) were compared between groups. Treatment effects within the predefined KRAS biomarker subsets were estimated by adjusted hazard ratio (HRadj) and 95% confidence interval (CI). EGFR mAb efficacy was measured within the KRAS wild-type subgroup according to BRAF and NRAS mutation status. In both KRAS wild-type and mutant subgroups, additional factors that could impact EGFR mAb efficacy were explored including the type of chemotherapy, line of therapy, age, sex, tumour sidedness and site of metastasis.Results5675 patients from 8 studies were included, all with known mCRC KRAS mutation status. OS (HRadj 0.90, 95% CI 0.84–0.98, p = 0.01) and PFS benefit (HRadj 0.73, 95% CI 0.68–0.79, p < 0.001) from EGFR mAbs was observed in the KRAS wild-type group. PFS benefit was seen in patients treated with fluorouracil (HRadj 0.75, 95% CI 0.68–0.82) but not with capecitabine-containing regimens (HRadj 1.04, 95% CI 0.86–1.26) (pinteraction = 0.002). Sidedness also interacted with EGFR mAb efficacy, with survival benefit restricted to left-sided disease (pinteraction = 0.038). PFS benefits differed according to age, with benefits greater in those under 70 (pinteraction = 0.001). The survival benefit was not demonstrated in those patients with mutations found in the KRAS, NRAS or BRAF genes. The presence of liver metastases interacted with EGFR mAb efficacy in patients with KRAS mutant mCRC (pinteraction = 0.004).ConclusionThe benefit provided by EGFR mAbs in KRAS WT mCRC is associated with left-sided primary tumour location, younger patient age and absence of NRAS or BRAF mutations. Survival benefit is observed with fluorouracil but not capecitabine. Exploratory results support further research in KRAS mutant mCRC without liver metastases.

Assessment of RAS-RAF-MAPK Pathway Mutation Status in Healthy Skin, Benign Nevi, and Cutaneous Melanomas: Pilot Study Using Droplet Digital PCR.

In the present study, we employed the ddPCR and IHC techniques to assess the prevalence and roles of RAS and RAF mutations in a small batch of melanoma (n = 22), benign moles (n = 15), and normal skin samples (n = 15). Mutational screening revealed the coexistence of BRAF and NRAS mutations in melanomas and nevi and the occurrence of NRAS G12/G13 variants in healthy skin. All investigated nevi had driver mutations in the BRAF or NRAS genes and elevated p16 protein expression, indicating cell cycle arrest despite an increased mutational burden. BRAF V600 mutations were identified in 54% of melanomas, and NRAS G12/G13 mutations in 50%. The BRAF mutations were associated with the Breslow index (BI) (p = 0.029) and TIL infiltration (p = 0.027), whereas the NRAS mutations correlated with the BI (p = 0.01) and the mitotic index (p = 0.04). Here, we demonstrate that the "young" ddPCR technology is as effective as a CE-IVD marked real-time PCR method for detecting BRAF V600 hotspot mutations in tumor biopsies and recommend it for extended use in clinical settings. Moreover, ddPCR was able to detect low-frequency hotspot mutations, such as NRAS G12/G13, in our tissue specimens, which makes it a promising tool for investigating the mutational landscape of sun-damaged skin, benign nevi, and melanomas in more extensive clinical studies.

Baseline ctDNA gene alterations as a biomarker of survival after panitumumab and chemotherapy in metastatic colorectal cancer.

Certain genetic alterations and right-sided primary tumor location are associated with resistance to anti-epidermal growth factor (EGFR) treatment in metastatic colorectal cancer (mCRC). The phase 3 PARADIGM trial (n = 802) demonstrated longer overall survival with first-line anti-EGFR (panitumumab) versus antivascular endothelial growth factor (bevacizumab) plus modified FOLFOX6 in patients with RAS wild-type mCRC with left-sided primary tumors. This prespecified exploratory biomarker analysis of PARADIGM (n = 733) evaluated the association between circulating tumor DNA (ctDNA) gene alterations and efficacy outcomes, focusing on a broad panel of gene alterations associated with resistance to EGFR inhibition, including KRAS, NRAS, PTEN and extracellular domain EGFR mutations, HER2 and MET amplifications, and ALK, RET and NTRK1 fusions. Overall survival was prolonged with panitumumab plus modified FOLFOX6 versus bevacizumab plus modified FOLFOX6 in patients with ctDNA that lacked gene alterations in the panel (that is, negative hyperselected; median in the overall population: 40.7 versus 34.4 months; hazard ratio, 0.76; 95% confidence interval, 0.62-0.92) but was similar or inferior with panitumumab in patients with ctDNA that contained any gene alteration in the panel (19.2 versus 22.2 months; hazard ratio, 1.13; 95% confidence interval, 0.83-1.53), regardless of tumor sidedness. Negative hyperselection using ctDNA may guide optimal treatment selection in patients with mCRC. ClinicalTrials.gov registrations: NCT02394834 and NCT02394795 .

Molecular screening with liquid biopsy for anti-EGFR retreatment in metastatic colorectal cancer: preliminary data from the randomized phase 2 PARERE trial.

Retreatment with anti-EGFR monoclonal antibodies is a promising strategy in patients with RAS/BRAF wild-type (wt) metastatic colorectal cancer (mCRC) who achieved benefit from previous anti-EGFR exposure upon exclusion of mutations in RAS/BRAF genes according to circulating tumor DNA (ctDNA) analysis by means of liquid biopsy (LB). This treatment approach is now being investigated in the randomized phase II trial PARERE (NCT04787341). We here present preliminary findings of molecular screening. Patients with RAS/BRAFV600E wt mCRC according to tissue genotyping who benefited from previous anti-EGFR-based treatment (fluoropyrimidines, oxaliplatin, irinotecan, and antiangiogenics) and then experienced disease progression to EGFR targeting were eligible for screening in the PARERE trial. The next-generation sequencing (NGS) panel Oncomine™ was employed for ctDNA testing. A total of 218 patients underwent LB, and ctDNA sequencing was successful in 201 of them (92%). RAS/BRAFV600E mutations were found in 68 (34%) patients and were mainly subclonal (median variant allele fraction [VAF] for KRAS, NRAS, and BRAF mutant clones: 0.52%, 0.62%, and 0.12%, respectively; p = 0.01), with KRASQ61H being the most frequently detected (31%). Anti-EGFR-free intervals did not predict ctDNA molecular status (p = 0.12). Among the 133 patients with RAS/BRAFV600E wt tumors according to LB, 40 (30%) harbored a mutation in at least another gene potentially implied in anti-EGFR resistance, mainly with subclonal expression (median VAF, 0.56%). In detail, alterations in PIK3CA, FBXW7, GNAS, MAP2K, ERBB2, BRAF (class I and II non-BRAFV600E), SMAD, EGFR, AKT1, and CTNNB1 occurred in 13%, 8%, 7%, 3%, 2%, 2%, 1%, 1%, 1%, and 1% cases, respectively. Co-mutations were detected in 13 (33%) out of 40 patients. This is the largest prospective cohort of mCRC patients screened with LB for anti-EGFR retreatment in a randomized study. ctDNA genotyping reveals that at least one out of three patients candidate for retreatment should be excluded from this therapy, and other potential drivers of anti-EGFR resistance are found in approximately one out of three patients with RAS/BRAFV600E wt ctDNA.

(245) Surveying the Genomic Landscape of Peyronie's Disease: A Study of 22 Cases

Abstract Introduction Since its description in 1743 by François de la Peyronie, penile fibromatosis continues to present an elusive pathogenesis. One of its distinguishing characteristics is the excessive proliferation of fibrogenic myofibroblasts, resulting in the formation of dense fibrous plaques that disrupt the normal cytoarchitecture of the tunica albuginea, giving rise to penile abnormalities. The possible clonal nature of the disease has been investigated by many studies and some have identified occasional karyotypic abnormalities, most commonly involving chromosomes 7, 8, 17, 18, X and Y (Mulhal JP et al 2004). Objective This study aimed to comprehensively analyze the genomic landscape of Peyronie's disease in a significant number of cases using a whole genome copy number variation assay (CNV). The objective was to identify any recurrent chromosomal gains or losses that could shed light on the pathogenesis of this disease, including the potential identification of an underlying clonal process. Methods Thirty-nine surgical specimens were retrospectively examined from men diagnosed with Peyronie's disease, collected between 1992 and 2022 (median age: 56 years, range: 17-73). The study was conducted under the approval of the Institutional Review Board (IRB # 22-006924). Tissue samples were fixed in 10% buffered formalin, embedded in paraffin, and subsequently subjected to histologic review and DNA analysis. DNA extraction followed standard protocols. Among the samples, 22 contained high-quality material suitable for CNV array analysis using the OncoScan SNV array, which encompassed approximately 900 genes/loci with a resolution of 50-125 kb. Additionally, the array included a somatic mutation panel targeting nine cancer-related genes. Results No significant loss or gain of chromosomal material was identified in the twenty-two cases analyzed. However, the somatic mutation panel revealed potential unconfirmed DNA mutations in NRAS, EGFR, and TP53. Ongoing studies are being conducted to confirm these mutations. Conclusions The etiology and pathogenesis of Peyronie's disease remain unresolved, presenting a challenging area of research. However, the findings of this study provide evidence that major recurrent losses or gains of chromosomal material are unlikely to play a significant role in the pathogenesis of this disease. While shedding light on this aspect, it is crucial to acknowledge that further research is necessary to confirm and expand upon these initial observations. Disclosure No.

GOLGA7 is essential for NRAS trafficking from the Golgi to the plasma membrane but not for its palmitoylation.

NRAS mutations are most frequently observed in hematological malignancies and are also common in some solid tumors such as melanoma and colon cancer. Despite its pivotal role in oncogenesis, no effective therapies targeting NRAS has been developed. Targeting NRAS localization to the plasma membrane (PM) is a promising strategy for cancer therapy, as its signaling requires PM localization. However, the process governing NRAS translocation from the Golgi apparatus to the PM after lipid modification remains elusive. This study identifies GOLGA7 as a crucial factor controlling NRAS' PM translocation, demonstrating that its depletion blocks NRAS, but not HRAS, KRAS4A and KRAS4B, translocating to PM. GOLGA7 is known to stabilize the palmitoyltransferase ZDHHC9 for NRAS and HRAS palmitoylation, but we found that GOLGA7 depletion does not affect NRAS' palmitoylation level. Further studies show that loss of GOLGA7 disrupts NRAS anterograde trafficking, leading to its cis-Golgi accumulation. Remarkably, depleting GOLGA7 effectively inhibits cell proliferation in multiple NRAS-mutant cancer cell lines and attenuates NRASG12D-induced oncogenic transformation in vivo. These findings elucidate a specific intracellular trafficking route for NRAS under GOLGA7 regulation, highlighting GOLGA7 as a promising therapeutic target for NRAS-driven cancers.

Association of KRAS G12C Status with Age at Onset of Metastatic Colorectal Cancer.

The association of age at the onset of CRC and the prevalence of a KRAS G12C mutation is unclear. A retrospective, multicenter study evaluating metastatic CRC patients from January 2019 to July 2023, treated at the Oncoclinicas units and tested for tissue based KRAS/NRAS and BRAF mutations in a centralized genomics lab. A mismatch repair (MMR) status was retrieved from different labs and electronic medical records, as were patient demographics (age, gender) and tumor sidedness. The chi-square test was used to examine the association between clinical and molecular variables, with p value < 0.05 being statistically significant. A total of 858 cases were included. The median age was 63.7 years (range 22-95) and 17.4% were less than 50 years old at the diagnosis of metastatic CRC. Male patients represented 50.3% of the population. The sidedness distribution was as follows: left side 59.2%, right side 36.8% and not specified 4%. The prevalence of the KRAS mutation was 49.4% and the NRAS mutation was 3.9%. Among KRAS mutated tumors, the most common variants were G12V (27.6%) and G12D (23.5%), while KRAS G12C was less frequent (6.4%), which represented 3.1% of the overall population. The BRAF mutant cases were 7.3% and most commonly V600E. Only five (<1%) non-V600E mutations were detected. MSI-high or dMMR was present in 14 cases (1.6%). In the age-stratified analysis, left-sidedness (p < 0.001) and a KRAS G12C mutation (p = 0.046) were associated with a younger age (<50 years). In the sidedness-stratified analysis, a BRAF mutation (p = 0.001) and MSI-high/dMMR status (p = 0.009) were more common in right-sided tumors. Our data suggest that KRAS G12C mutations are more frequent in early-onset metastatic CRC. To the best of our knowledge, this is the largest cohort in the Latin American population with metastatic CRC reporting RAS, BRAF and MSI/MMR status.

KRAS, NRAS and BRAF Mutational Profile of Colorectal Cancer in a Series of Moroccan Patients.

The present study aimed to evaluate the frequencies of KRAS, NRAS, and BRAF mutations and their possible associations with clinicopathological features in 249 Moroccan patients with colorectal cancer (CRC). A retrospective investigation of a cohort of formalin-fixed paraffin-embedded tissues of 249 patients with CRC was screened for KRAS/NRAS/BRAF mutations using Idylla™ technology and pyrosequencing. KRAS, NRAS, and BRAF mutations were revealed in 46.6% (116/249), 5.6% (14/249), and 2.4% (6/249) of patients. KRAS exon 2 mutations were identified in 87.9% of patients (102/116). KRAS G12D and G12C were the most frequent, at 32.8% and 12.93%, respectively. Among the patients with KRAS exon 2 wild-type (wt), 27.6% (32/116) harbored additional KRAS mutations. Concurrent KRAS mutations were identified in 9.5% (11/116); including six in codon 146 (A146P/T/V), three in codon 61 (Q61H/L/R), one in codon 12 (G12A and Q61H), and one in codon 13 (G13D and Q61L). Among the NRAS exon 2wt patients, 64.3% (9/14) harbored additional NRAS mutations. Concurrent NRAS mutations were identified in 28.6% (4/14) of NRAS-mutant patients. Since 3.2% wt KRAS were identified with NRAS mutations, concomitant KRAS and NRAS mutations were identified in 2.4% (6/249) of patients. KRAS mutations were higher in the >50-year-old age-group (P = .031), and the tumor location was revealed to be significantly associated with KRAS mutations (P = .028) predominantly in left colon (27.5%) and colon (42.2%) locations. NRAS mutations were most prevalent in the left colon (42.8%) and in well-differentiated tumors (64.2%). Detection of KRAS mutations, particularly the G12C subtype, may be significant for patients with CRC and has possible therapeutic implications. However, rare KRAS concomitant mutations in CRC patients suggest that each individual may present distinct therapeutic responses. KRAS testing alongside the identification of other affected genes in the same patient will make the treatments even more personalized by contributing more accurately to the clinical decision process. Overall, early diagnosis using novel molecular techniques may improve the management of CRC by providing the most efficient therapies for Moroccan patients.

NRAS Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth.

Our findings suggest that targeting the AHCYL1-IP3R axis presents a novel therapeutic approach for NRAS-mutated melanomas, with potential applicability to all cancers harboring RAS mutations, such as KRAS-mutated human colorectal cancers.

The Effect of Non-Overlapping Somatic Mutations in BRAF, NRAS, NF1, or CKIT on the Incidence and Outcome of Brain Metastases during Immune Checkpoint Inhibitor Therapy of Metastatic Melanoma

The Effect of Non-Overlapping Somatic Mutations in BRAF, NRAS, NF1, or CKIT on the Incidence and Outcome of Brain Metastases during Immune Checkpoint Inhibitor Therapy of Metastatic Melanoma

Uveal melanoma: Recent advances in immunotherapy.

Uveal melanoma (UM) is the most common primary intraocular cancer in adults. The incidence in Europe and the United States is 6-7 per million population per year. Although most primary UMs can be successfully treated and locally controlled by irradiation therapy or local tumor resection, up to 50% of UM patients develop metastases that usually involve the liver and are fatal within 1 year. To date, chemotherapy and targeted treatments only obtain minimal responses in patients with metastatic UM, which is still characterized by poor prognosis. No standard therapeutic approaches for its prevention or treatment have been established. The application of immunotherapy agents, such as immune checkpoint inhibitors that are effective in cutaneous melanoma, has shown limited effects in the treatment of ocular disease. This is due to UM's distinct genetics, natural history, and complex interaction with the immune system. Unlike cutaneous melanomas characterized mainly by BRAF or NRAS mutations, UMs are usually triggered by a mutation in GNAQ or GNA11. As a result, more effective immunotherapeutic approaches, such as cancer vaccines, adoptive cell transfer, and other new molecules are currently being studied. In this review, we examine novel immunotherapeutic strategies in clinical and preclinical studies and highlight the latest insight in immunotherapy and the development of tailored treatment of UM.

GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).

Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy

BackgroundMEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib.MethodsThe effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi.ResultsDiethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib.ConclusionsDisulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

Missense mutation of NRAS is associated with malignant progression in neurocutaneous melanosis

Neurocutaneous melanosis (NCM) is a rare congenital neurocutaneous syndrome characterized by congenital melanocytic nevus of skin and abnormal proliferation of leptomeningeal melanocytes. Early acquisition of post-zygotic somatic mutations has been postulated to underlie the pathogenesis of NCM. The pathogenesis of NCM remains to be fully elucidated, and treatment options have not been established. Here, we report for the first time, multiregional genomic analyses in a 3-year-old autopsied girl with leptomeningeal melanomatosis associated with NCM, in which a ventriculo-peritoneal (VP) shunt was inserted for the treatment of hydrocephalus. The patient expired six months after the onset due to respiratory failure caused by abdominal dissemination via VP shunt. We performed multiregional exome sequencing to identify genomic differences among brain and abdominal tumors, nevus, and normal tissues. A total of 87 somatic mutations were found in 71 genes, with a significantly large number of gene mutations found in the tumor site. The genetic alterations detected in the nevus were only few and not shared with other sites. Three mutations, namely GNAQ R183Q, S1PR3 G89S and NRAS G12V, considered pathogenic, were found, although S1PR3 mutations have not been previously reported in melanocytic tumors. GNAQ and S1PR3 mutations were shared in both tumor and normal sites. Moreover, the mutant allele frequencies of the two mutations were markedly higher in tumor sites than in normal sites, with copy-neutral loss-of-heterozygosity (CN-LOH) occurring in tumor. NRAS mutation was found only in the abdominal tumor and was thought to be responsible for malignant progression in the present case. Multiregional comprehensive genetic analysis may lead to discovering novel driver mutations associated with tumorigenesis and targeted therapy.