ARAF mutations confer resistance to the RAF inhibitor belvarafenib in melanoma.

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

Exploring Molecular Genetic Alterations and RAF Fusions in Melanoma: A Belvarafenib Expanded Access Program in Patients with RAS/RAF-Mutant Melanoma.

BACKGROUND: Melanoma incidence is on the rise in East Asia, yet studies of the molecular landscape are lacking in this population. We examined patients with melanoma who underwent next-generation sequencing (NGS) at a single tertiary center in South Korea, focusing on patients harboring NRAS or RAF alterations who received belvarafenib, a pan-RAF dimer inhibitor, through the Expanded Access Program (EAP). PATIENTS AND METHODS: Data were collected from 192 patients with melanoma who underwent NGS between November 2017 and May 2023. Variant call format data were obtained and annotated. Patients in the EAP received 450 mg twice daily doses of belvarafenib. RESULTS: Alterations in the RAS/RTK pathway were the most prevalent, with BRAF and NRAS alteration rates of 22.4% and 17.7%, respectively. NGS enabled additional detection of fusion mutations, including 6 BRAF and 1 RAF1 fusion. Sixteen patients with NRAS or RAF alterations received belvarafenib through the EAP, and disease control was observed in 50%, with 2 patients demonstrating remarkable responses. CONCLUSIONS: Our study highlights the value of NGS in detecting BRAF, NRAS mutations and RAF fusions, expanding possibilities for targeted therapies in malignant melanoma. Belvarafenib showed clinical benefit in patients harboring these alterations. Ongoing trials will provide further insights into the safety and efficacy of belvarafenib.

Quantitative assessments of pupillary light reflexes in hospital-onset unresponsiveness.

BACKGROUND: Patients who develop hospital-onset unresponsiveness should be promptly managed in order to avoid clinical deterioration. Pupillary examination through pupillary light reflex is the gold standard method in the initial evaluation of unresponsive patients. However, the current method of shining light and subjective description often shows poor reliability. The objective of this study is to explore whether a quantitative measurement of pupillary light reflexes is useful in detecting brain herniation syndrome and predicting neurological outcomes in patients who developed hospital-onset unresponsiveness after admission for non-neurological reasons. METHODS: This was a registry-based observational study on patients who activated the neurological rapid response team at Asan Medical Center (Seoul, Korea). Hospital-onset unresponsiveness was defined as a newly developed unresponsive state as assessed by the ACDU (Alert, Confused, Drowsy, and Unresponsive) scale during the hospital stay. Demographics, comorbidities, pupillometry parameters including Neurological Pupil index, brain herniation syndrome, in-hospital mortality, and modified Rankin Scale at 3-months were analyzed. RESULTS: In 214 consecutive patients with hospital-onset unresponsiveness, 37 (17%) had brain herniation syndrome. The optimal cut-off value of Neurological Pupil index for detecting brain herniation syndrome was < 1.6 (specificity, 91% [95% confidence interval (CI) = 86-95]; sensitivity, 49% [95% CI = 32-66]). The in-hospital mortality rate was 28% (59/214); the Neurological Pupil index was negatively associated with in-hospital mortality after adjustments for the presence of brain herniation syndrome (adjusted odds ratio = 0.77, 95% CI = 0.62-0.96). Poor neurological outcomes (modified Rankin Scale ≥4) at 3 months was observed in 76% (152/201) of the patients; the Neurological Pupil index was negatively associated with poor neurological outcomes after adjustments for clinical variables (adjusted odds ratio = 0.67, 95% CI = 0.49-0.90). CONCLUSIONS: Quantitative measurements of pupillary light reflexes may be useful for early detection of potentially life-threatening neurological conditions in patients with hospital-onset unresponsiveness.

Hemodynamic contribution of transdural collateral flow in adult patients with moyamoya disease.

To evaluate the hemodynamic contributions of collateral flow in adult patients with moyamoya disease, neurological deterioration or fluctuation during admission, Suzuki grade, various collateral routes, lesion volume, cerebral blood flow (CBF), and their associations were analyzed. Thirty patients (60 cerebral hemispheres, mean age 45 ± 25 years, and 73.3 % female) who were diagnosed with moyamoya disease or syndrome were enrolled over 3 years. Moyamoya stages from each hemisphere were stratified according to the Suzuki's criteria through six-vessel angiography into internal carotid arteries (ICAs), external carotid arteries (ECAs), and vertebral arteries (VAs). Collateral routes were categorized into the circle of Willis, leptomeningeal, and transdural. The volume of ipsilateral infarction was analyzed by magnetic resonance imaging. CBF volume was measured using color-coded duplex sonography. Suzuki's grade was inversely correlated with flow volume of the ICAs (p < 0.001), whereas no association was found with that of the ECAs (p = 0.445) or VAs (p = 0.096). Among hemispheres with ≥ grade 3 (n = 36), patients with transdural ECA collateral flow had less neurological deterioration or fluctuation (0.0 vs. 30.8 %, p = 0.047), smaller lesion volume (2.4 ± 3.6 vs. 27.6 ± 59.3 mL, p = 0.041), lower ICA flow (88.4 ± 45.9 vs. 146.2 ± 121.7 mL/min, p = 0.022), higher ECA flow (205.7 ± 77.7 vs. 135.9 ± 52.7 mL/min, p = 0.046), and a higher ECA/ICA flow volume ratio (31.8 ± 92.8 vs. 1.7 ± 1.9, p = 0.024). Our results suggest that ICA flow volume is inversely correlated with Suzuki grade, and that transdural ECA collaterals appear to be an important detour in adult patients with advanced stage moyamoya disease, suggesting a protector against an impending ischemic attack.

Encephalopathy After a High-Dose Dexamethasone Suppression Test in a Woman With X-Linked Ornithine Transcarbamylase Deficiency.

Background/Objective: The high-dose dexamethasone suppression test is a common and usually benign endocrine procedure. We report a patient with ornithine transcarbamylase deficiency (OTCD) who developed hyperammonemic encephalopathy after a high-dose dexamethasone suppression test. Case Report: A 46-year-old woman with a 1.3-cm right adrenal incidentaloma causing mild autonomous cortisol secretion underwent a high-dose dexamethasone suppression test for confirming adrenocorticotropic hormone independency. On the next day, she presented to the emergency room with confusion and somnolence. Her Glasgow Coma Scale score was 10 on arrival. The initial laboratory results showed ammonia, alanine transaminase, creatinine, and blood urea nitrogen levels of 289.51 (18.73-54.5) µg/dL, 21 (≤33) IU/L, 0.6 (0.6-1.1) mg/dL, and 13 (7-20) mg/dL, respectively. Electroencephalography showed triphasic morphology with no pathologies on brain imaging. Her husband told us that her brother and son had died in the neonatal period. On further review of medical records, we found that she was diagnosed as an OTCD carrier. We administered L-arginine, L-carnitine, rifaximin, and continuous renal replacement therapy. After 3 days, the serum ammonia level was 78.34 µg/dL with an increased Glasgow Coma Scale score of 15, and electroencephalography abnormalities disappeared. Discussion: Liver diseases and urea cycle disorders are the leading causes of hyperammonemia. This causes encephalopathy and death if the ammonia levels are too high. X-linked OTCD urea cycle disorder affects men more severely as they have only the carrier X chromosome. Glucocorticoids can exacerbate this disorder because they increase protein substrates converted to ammonia. Conclusion: This case reminds that it may be particularly important to have a complete medical history when administering glucocorticoids.

Avelumab first-line maintenance treatment in patients with locally advanced or metastatic urothelial carcinoma: real-world results from a Korean expanded access program.

Background: The JAVELIN Bladder 100 phase 3 trial demonstrated the efficacy and safety of avelumab administered as first-line (1L) maintenance treatment in patients with advanced urothelial carcinoma (UC) without disease progression after 1L platinum-based chemotherapy. This study provides the first real-world data from Korea regarding avelumab 1L maintenance treatment, comprising data obtained from a nationwide expanded access program (EAP). Methods: This open-label EAP was conducted at five centers from September 2021 until June 2023. Eligible patients had unresectable locally advanced or metastatic UC and were progression free after 1L platinum-based chemotherapy. Patients received avelumab 10 mg/kg intravenously every 2 weeks per local prescribing information. Safety and effectiveness were assessed by treating physicians according to routine practice. Results: Overall, 30 patients were enrolled. At initial UC diagnosis, 20 patients (66.7%) had stage 4 disease and 12 (40.0%) had visceral metastases. The most common 1L chemotherapy regimen was gemcitabine + cisplatin (21 patients; 70.0%). All but one patient (96.7%) had received 4-6 cycles of 1L chemotherapy. The median interval from end of 1L chemotherapy to start of avelumab was 4.4 weeks. Median duration of avelumab treatment was 6.2 months (range, 0.9-20.7); nine patients (30.0%) received >12 months of treatment. Adverse events related to avelumab occurred in 21 patients (70.0%) and were grade ≥3 or classified as serious in three patients (10.0%). Median progression-free survival was 7.9 months (95% CI, 4.3-13.1). Overall survival was not analyzed because only one patient died. Conclusion: Results from this EAP demonstrated the clinical activity and acceptable safety of avelumab 1L maintenance treatment in Korean patients with advanced UC, consistent with previous studies.

Metabolite investigation in both anterior and posterior cingulate gyri in Alzheimer's disease spectrum using 3-tesla MR spectroscopy.

AIMS: In order to evaluate the metabolite changes of both anterior and posterior cingulate gyri during the progression of Alzheimer's disease (AD) pathology, a 3-tesla MR spectroscopy study was performed. METHODS: Thirty-six patients with AD, 19 patients with amnestic mild cognitive impairment (aMCI), and 23 cognitively normal (CN) subjects were recruited. MR spectroscopy was conducted within the anterior and posterior cingulate gyri. A one-way analysis of co-variance was used to compare the metabolite ratios of each group and correlation analysis was used to show the correlation between the metabolite ratios with the Mini-Mental State Examination (MMSE) and Neuropsychiatric Inventory (NPI). RESULTS: The N-acetylaspartate/creatine (NAA/Cr) of the posterior cingulate gyrus was significantly higher in CN subjects than in aMCI and AD patients. On the other hand, the myoinositol/creatine (ml/Cr) of the anterior cingulate gyrus was significantly higher in AD patients than in CN subjects and aMCI patients. The ml/Cr of the posterior cingulate gyrus correlated with the MMSE and that of the anterior cingulate gyrus correlated with the NPI. CONCLUSION: Both the decreased NAA/Cr of the posterior cingulate gyrus and the increased ml/Cr of the anterior cingulate gyrus may reflect biochemical changes in AD according to the posterior-dominant progression of AD pathology.

Functional investigation of bilateral posterior cingulate gyri using multivoxel MR spectroscopy.

The exact functional correlation of each hemisphere's posterior cingulate gyrus with the symptoms of Alzheimer's disease (AD) remains unknown. We attempted to evaluate the relationship between metabolite ratios in each hemisphere's posterior cingulate gyrus and cognitive deficits, using multivoxel magnetic resonance spectroscopy (MRS). We recruited 23 patients with AD, 16 patients with amnestic mild cognitive impairment and 22 cognitively normal subjects. All patients underwent multivoxel MRS in the bilateral posterior cingulate gyri. We statistically analyzed correlations between the N-acetylaspartate/creatine ratio (NAA/Cr) in each posterior cingulate gyrus and patients' raw scores on neuropsychological tests. The NAA/Cr of each posterior cingulate gyrus correlated well with the verbal learning test scores on immediate recall and delayed recall tasks. We found that the only cognitive domain to correlate with the NAA/Cr of each posterior cingulate gyrus was verbal memory. Our results did not show any significant functional difference between right and left posterior cingulate gyri.

Neurological and clinical status from early time point to long-term follow-up after in-hospital cardiac arrest.

AIM: We aimed to evaluate neurological profiles of patients with in-hospital cardiac arrest (IHCA) from early time points to long-term follow-up periods. METHODS: For this prospective cohort study, we established a neurological rapid response team, and serially evaluated the neurological status of patients with IHCA from the initial resuscitation to 12 months after the onset of IHCA. The primary outcome was good neurological status defined as a Clinical Performance Category score of 1-2 at 12 months after IHCA. The secondary outcomes included the awakening and neurological recovery during the first week, the survival and neurological status at hospital discharge, and the survival at 12 months. RESULTS: A total of 291 adult patients with IHCA were included. On the first day and during the first week after IHCA, the awakening was achieved in 61 (21.0 %) and 119 patients (40.9 %), respectively; and neurological recovery in 12 (4.1 %) and 46 patients (15.8 %), respectively. Epileptic seizures developed in 9.7 % following restoration of spontaneous circulation. At hospital discharge, 106 patients (36.4 %) had survived; among them, 63.2 % showed good neurological status. At 12 months, 63 (21.6 %) patients survived; among them, 81.7 % showed good neurological status (17.0 % among all patients with IHCA). Of patients without awakening during the first 3 and 7 days, 2.7 % and 1.2 % showed good neurological status at 12 months, respectively. CONCLUSIONS: Among patients with IHCA, awakening and neurological recovery were remarkable throughout the first week. Survival and good neurological status were substantial at 12 months after IHCA.

Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals.

In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1% (33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage.

Flavonoids from the flower of Rhododendron yedoense var. poukhanense and their antioxidant activities.

Antioxidant flavonoids have been isolated from the flower of Rhododendron yedoense var. poukhanense. One new flavonoid and three known flavonoids, quercetin-5-O-beta-D-glucopyranoside (1), quercetin (3), and quercitrin (4), were isolated from the butanol and ethyl acetate extracts of the plant. The new flavonoid was identified as myricitrin-5-methyl ether (2). The isolation of these flavonoids from this plant, for the first time, is a valuable finding. The flavonoids were evaluated for their antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH), TBARS (thiobarbituric acid reactive substance) and superoxide anion radical (O2-) in the xanthine/xanthine oxidase assay system. In the DPPH scavenging assay, the IC50 values were 4.5 +/- 0.48 microM for compound 2 and 9.7 +/- 0.29 microM for compound 3, which showed an antioxidant activity approximately 1.5-2 times higher than the antioxidant activity of alpha-tocopherol (9.8 +/- 0.94 microM). Additionally, the antioxidant activities of myricitrin-5-methyl ether (2) (IC50 = 1.7 +/- 0.22 microM) and quercetrin (4) (IC50 = 1.9 +/- 0.63 microM) were higher than that of L-ascorbic acid (IC50 = 7.4 +/- 0.63 microM) when evaluated using a TBARS assay. Compound 2 showed high activity in both the inhibition of xanthine oxidase (1.1 +/- 0.21 mM) and in the activation of superoxide scavenging.

A role for the carbohydrate portion of ginsenoside Rg3 in Na+ channel inhibition.

We showed recently that ginsenosides inhibit the activity of various types of ion channel. Here we have investigated the role of the carbohydrate component of ginsenoside Rg3 in the inhibition of Na+ channels. The channels were expressed in Xenopus oocytes by injecting cRNAs encoding rat brain Nav1.2 alpha and beta1 subunits, and analyzed by the two-electrode voltage clamp technique. Treatment with Rg3 reversibly inhibited the inward Na+ peak current (INa) with an IC50 of 32.2 +/- 4.5 microM, and the inhibition was voltage-dependent. To examine the role of the sugar moiety, we prepared a straight chain form of the second glucose and a conjugate of this glucose with 3-(4-hydroxyphenyl) propionic acid hydrazide (HPPH). Neither derivative inhibited INa. Treatment with the carbohydrate portion of ginsenoside Rg3, sophorose [beta-D-glucopyranosyl (1-->2)- beta-glucopyranoside], or the aglycone (protopanaxadiol), on their own or in combination had no effect on INa. These observations indicate that the carbohydrate portion of ginsenoside Rg3 plays an important role in its effect on the Na+ channel.

ROS Accumulation by PEITC Selectively Kills Ovarian Cancer Cells via UPR-Mediated Apoptosis.

Unfolded protein response (UPR) is crucial for both survival and death of mammalian cells, which is regulated by reactive oxygen species (ROS) and nutrient depletion. In this study, we demonstrated the effect of ROS-accumulation, induced by ß-phenethyl isothiocyanate (PEITC), on UPR-mediated apoptosis in ovarian cancer cells. We used ovarian cancer cell lines, PA-1 and SKOV-3, with different p53 status (wild- and null-type, respectively). PEITC caused increased ROS-accumulation and inhibited proliferation selectively in ovarian cancer cells, and glutathione (GSH) depletion in SKOV-3. However, PEITC did not cause any effect in normal ovarian epithelial cells and peripheral blood mononuclear cells. After 48 h of PEITC treatment (5 µM), apoptotic cell death was shown to increase significantly in the ovarian cancer cells and not in the normal cells. The key regulator of UPR-mediated apoptosis, CHOP/GADD153 and endoplasmic reticulum resident chaperone BiP/GRP78 were parallely up-regulated with activation of two major sensors of the UPR [PERK and ATF-6 in PA-1; PERK, and IRE1α in SKOV-3) in response to ROS accumulation induced by PEITC (5 µM). ROS scavenger, N-acetyl-L-cysteine (NAC), attenuated the effect of PEITC on UPR signatures (P-PERK, IRE1α, CHOP/GADD153, and BiP/GRP78), suggesting the involvement of ROS in UPR-mediated apoptosis. Altogether, PEITC induces UPR-mediated apoptosis in ovarian cancer cells via accumulation of ROS in a cancer-specific manner.

Ipsilateral visual illusion after unilateral posterior cerebral artery infarction: a report of two cases.

In cases of unilateral posterior cerebral artery (PCA) infarction, abnormal visual perception in the ipsilateral visual field, which is usually believed to be intact, is not met frequently and may confuse doctors during evaluation. Recently, we observed two patients who presented with contralateral hemianopsia accompanied by ipsilateral visual illusions after acute unilateral PCA infarctions. Their visual illusion was characterized by zooming in, macropsia or micropsia. These symptoms appeared to be related to deficits in size constancy. Lesions of both patients commonly involved the ipsilateral forceps major. The consistent presentation observed in these two patients suggests that dominance of size constancy can be located in the left hemisphere in some individuals.