SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

Stellate ganglion block to mitigate thalamic pain syndrome of an oncological origin.

BACKGROUND: Thalamic pain syndrome (TPS) is an enigmatic and rare condition. Thalamic pain syndrome is under the umbrella of central pain syndrome, which is classically associated with multiple sclerosis, spinal cord injury, postamputation, epilepsy, stroke, tumor, and Parkinson's disease. The mainstay treatment of TPS is polypharmacy. There is uncertainty about the intermediate options to manage medication-resistant TPS before resorting to invasive, and often expensive, intracranial therapies. Stellate ganglion block (SGB) has shown promise in reducing TPS symptoms of the upper extremity and face following a thalamic ischemic event. AIMS: Discuss the effect and potential utility of SGB on ipsilateral headache, facial, and upper extremity neuropathic pain due to thalamic malignancies. MATERIALS AND METHODS: A review of two patient records that underwent SGB for treatment of TPS of oncologic origin. RESULTS: We present two cases of the successful use of SGB for the treatment of oncologic-related TPS for patients who had failed other conservative pharmacologic measures. DISCUSSION: Chronic pain is a complex experience that often simultaneously involves psychosocial, neuropathic, and nociceptive constituents. Among advanced cancer patients, factors such as an individual's spirituality, psychological stressors, and views on their mortality add layers of intricacy in addressing their pain. While TPS has been characterized in both stroke populations and oncologic populations, the treatment of SGB for pain relief in TPS has been limited to the stroke population. Repeated SGB worked to alleviate the ipsilateral headache, facial, and upper extremity pain in these two patients. The benefits of utilization of SGB, with the possibility of pain relief, within the thalamic malignancy population cannot be understated. CONCLUSION: In summary, ultrasound-guided SGB may be considered in patients with TPS due to thalamic cancer, before pursuing more invasive intracranial surgeries to treat pain.

The PZP Domain of AF10 Senses Unmodified H3K27 to Regulate DOT1L-Mediated Methylation of H3K79.

AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism by which AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L.

A single-center, retrospective analysis to compare measurement of fibrinogen using the TEG6 analyzer to the Clauss measurement in children undergoing heart surgery.

BACKGROUND: Newer generation viscoelastic tests, TEG6s, offer point-of-care hemostatic therapy in adult patients. However, their efficacy in estimating fibrinogen levels in pediatric patients undergoing cardiac surgery is not well established. AIMS: This study evaluates TEG6s for estimating fibrinogen levels in pediatric cardiac surgery patients and its predictive capability for post-bypass hypofibrinogenemia. METHODS: A single-center, retrospective study on pediatric patients (under 18 years) who underwent cardiac surgery with cardiopulmonary bypass from August 2020 and November 2022. Blood samples for estimated whole blood functional fibrinogen level via TEG6s (Haemonetics Inc.) and concurrent laboratory-measured plasma fibrinogen via von Clauss assay were collected at pre- and post-cardiopulmonary bypass. RESULTS: Paired data for TEG6s estimated functional fibrinogen levels and plasma fibrinogen were analyzed for 432 pediatric patients pre-bypass. It was observed that functional fibrinogen consistently overestimated plasma fibrinogen across all age groups with a mean difference of 138 mg/dL (95% confidence interval [CI]: 128-149 mg/dL). This positive bias in the pre-bypass data was confirmed by Bland-Altman analysis. Post-bypass, functional fibrinogen estimates were comparable to plasma fibrinogen in all patient groups with a mean difference of -6 mg/dL (95% CI: -20-8 mg/dL) except for neonates, where functional fibrinogen levels underestimated plasma fibrinogen with a mean difference of -38 mg/dL (95% CI: -64 to -12 mg/dL). The predictive accuracy of functional fibrinogen for detecting post-bypass hypofibrinogenemia (plasma fibrinogen ≤250 mg/dL) demonstrated overall fair accuracy in all patients, indicated by an area under the curve of 0.73 (95% CI: 0.65-0.80) and good accuracy among infants, with an area under the curve of 0.80 (95% CI: 0.70-0.90). Similar performance was observed in predicting critical post-bypass hypofibrinogenemia (plasma fibrinogen ≤200 mg/dL). Based on these analyses, optimal cutoffs for predicting post-bypass hypofibrinogenemia were established as a functional fibrinogen level ≤270 mg/dL and MAFF ≤15 mm. CONCLUSION: This study demonstrates that whole blood functional fibrinogen, as estimated by TEG6s, tends to overestimate baseline plasma fibrinogen levels in pediatric age groups but aligns more accurately post-cardiopulmonary bypass, particularly in neonates and infants, suggesting its potential as a point-of-care tool in pediatric cardiac surgery. However, the variability in TEG6s performance before and after bypass highlights the need for careful interpretation of its results in clinical decision-making. Despite its contributions to understanding TEG6s in pediatric cardiac surgery, the study's design and inherent biases warrant cautious application of these findings in clinical settings.

Scavenger Receptors: Novel Roles in the Pathogenesis of Liver Inflammation and Cancer.

The scavenger receptor superfamily represents a highly diverse collection of evolutionarily-conserved receptors which are known to play key roles in host homeostasis, the most prominent of which is the clearance of unwanted endogenous macromolecules, such as oxidized low-density lipoproteins, from the systemic circulation. Members of this family have also been well characterized in their binding and internalization of a vast range of exogenous antigens and, consequently, are generally considered to be pattern recognition receptors, thus contributing to innate immunity. Several studies have implicated scavenger receptors in the pathophysiology of several inflammatory diseases, such as Alzheimer's and atherosclerosis. Hepatic resident cellular populations express a diverse complement of scavenger receptors in keeping with the liver's homeostatic functions, but there is gathering interest in the contribution of these receptors to hepatic inflammation and its complications. Here, we review the expression of scavenger receptors in the liver, their functionality in liver homeostasis, and their role in inflammatory liver disease and cancer.

The environmental impact of inhalers for asthma: A green challenge and a golden opportunity.

The propellants in metered-dose inhalers (MDIs) are powerful greenhouse gases, which account for approximately 13% of the NHS's carbon footprint related to the delivery of care. Most MDI use is in salbutamol relievers in patients with poorly controlled disease. The UK lags behind Europe in this regard, with greater reliance on salbutamol MDI and correspondingly greater greenhouse gas emissions, roughly treble that of our European neighbours. There has been a broad switch towards MDIs in asthma treatment in the UK over the last 20 years to reduce financial costs, such that the treatment for two-thirds of asthma patients in the UK is dominated by salbutamol MDI. Strategies that replace overuse of reliever MDIs with regimes emphasising inhaled corticosteroids have the potential to improve asthma control alongside significant reductions in greenhouse gas emissions. Real-world evidence shows that once-daily long-acting combination dry-powder inhalers (DPIs) can improve compliance and asthma control, and reduce the carbon footprint of care. Similarly, maintenance and reliever therapy (MART), which uses combination reliever and inhaled steroids in one device (usually a DPI), can simplify therapy, improve asthma control and reduce greenhouse gas emissions. Both treatment strategies are popular with patients, most of whom would be willing to change treatment to reduce their carbon footprint. By focussing on patients who are currently using high amounts of salbutamol MDI and prioritising inhaled steroids via DPIs, there are golden opportunities to make asthma care in the UK more effective, safer and greener.

An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation.

Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N3 methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases-human SETD6 and the plant LSMT-but differs in the residues constituting the active site. In the SETD3 active site, Asn255 engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (kcat/Km ) on histidine than on lysine. Here, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn255 with phenylalanine (N255F), together with substitution of Trp273 with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms-the N3 atom of histidine and the terminal ϵ-amino nitrogen of lysine-occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn255 substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.

Characterization of SETD3 methyltransferase-mediated protein methionine methylation.

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain-containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr312 and Trp273, as well as the hydrocarbon side chain of Ile310 Our results suggest that placing methionine properly in the active site-within close proximity to and in line with the incoming methyl group of SAM-would allow some SET domain proteins to selectively methylate methionine in proteins.

Downregulation of the αvß6 Integrin via RGD Engagement Is Affinity and Time Dependent.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvß6) has been identified as playing a key role in the activation of transforming growth factor-ß (TGFß) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvß6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGFß inhibition. High αvß6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvß6 affinity, slow dissociation) and SC-68448 (low αvß6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvß6 in normal human bronchial epithelial (NHBE) cells. After washout, the αvß6 cell surface repopulation was faster for SC-68448 compared with compound 1 In addition, αvß6-dependent release of active TGFß from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFß was restored, whereas after washout of compound 1 the inhibition of TGFß activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvß6 in NHBE cells if present continuously. These observations suggest αvß6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvß6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation. SIGNIFICANCE STATEMENT: The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvß6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvß6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvß6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.

SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

A molecular wound response program associated with regeneration initiation in planarians.

Planarians are capable of regenerating any missing body part and present an attractive system for molecular investigation of regeneration initiation. The gene activation program that occurs at planarian wounds to coordinate regenerative responses remains unknown. We identified a large set of wound-induced genes during regeneration initiation in planarians. Two waves of wound-induced gene expression occurred in differentiated tissues. The first wave includes conserved immediate early genes. Many second-wave genes encode conserved patterning factors required for proper regeneration. Genes of both classes were generally induced by wounding, indicating that a common initial gene expression program is triggered regardless of missing tissue identity. Planarian regeneration uses a population of regenerative cells (neoblasts), including pluripotent stem cells. A class of wound-induced genes was activated directly within neoblasts, including the Runx transcription factor-encoding runt-1 gene. runt-1 was required for specifying different cell types during regeneration, promoting heterogeneity in neoblasts near wounds. Wound-induced gene expression in neoblasts, including that of runt-1, required SRF (serum response factor) and sos-1. Taken together, these data connect wound sensation to the activation of specific cell type regeneration programs in neoblasts. Most planarian wound-induced genes are conserved across metazoans, and identified genes and mechanisms should be important broadly for understanding wound signaling and regeneration initiation.

A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin.

The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2.

Histone-binding domains: strategies for discovery and characterization.

Chromatin signaling dynamics fundamentally regulate eukaryotic genomes. The reversible covalent post-translational modification (PTM) of histone proteins by chemical moieties such as phosphate, acetyl and methyl groups constitutes one of the primary chromatin signaling mechanisms. Modular protein domains present within chromatin-regulatory activities recognize or "read" specifically modified histone species and transduce these modified species into distinct downstream biological outcomes. Thus, understanding the molecular basis underlying PTM-mediated signaling at chromatin requires knowledge of both the modification and the partnering reader domains. Over the last ten years, a number of innovative approaches have been developed and employed to discover reader domain binding events with histones. Together, these studies have provided crucial insight into how chromatin pathways influence key cellular programs. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

Thoughtful prescription of inhaled medication has the potential to reduce inhaler-related greenhouse gas emissions by 85.

INTRODUCTION: Both physicians and patients are increasingly aware of the environmental impacts of medication. The shift of treatment paradigm towards MART-treatment (Maintenance and Reliever Therapy) in asthma affects the treatment-related emissions. The carbon footprint of inhaled medication is also tied to the type of the device used. Today the most commonly used propellant-containing pressurised metered-dose inhalers (pMDIs) have a carbon footprint typically 20-40-fold higher than propellant-free dry powder inhalers (DPIs) and soft mist inhalers. METHODS: We analysed the carbon footprint of inhaled medications in Europe using published life cycle analyses of marketed inhalers and comprehensive 2020 European sales data. In addition, we give an estimate on treatment-related emissions of different treatment regimens on Global Initiative for Asthma (GINA) step 2. RESULTS: There is potential to reduce the carbon footprint of inhaled medications by 85% if DPIs are preferred over pMDIs. Emissions from pMDIs in the EU were estimated to be 4.0 megatons of carbon dioxide equivalent (MT CO2e) and this could be reduced to 0.6 MT CO2e if DPIs were used instead. In the treatment of moderate asthma with DPI, an as-needed combination of inhaled corticosteroid and long-acting beta-agonist in a single inhaler had a substantially lower annual carbon footprint (0.8 kg CO2e) than the more traditional maintenance therapy with an inhaled corticosteroid alone with as-needed short-acting beta-agonist (2.9 kg CO2e). DISCUSSION: There has been an urgent call for healthcare to reduce its carbon footprint for appropriate patients with asthma and chronic obstructive pulmonary disease (COPD), changing to non-propellant inhalers can reduce the carbon footprint of their treatment by almost 20-fold.

Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Genicular nerve neurolysis with phenol for chronic knee pain: A case series.

Chronic knee pain continues to be a common complaint amongst patients and continues to grow as the elderly population lives longer. New ways to treat chronic pain conditions, including chronic knee pain, are necessary as increased co-morbidities prevent patients from being surgical candidates. Recently, the genicular nerves have received more attention due to their role in innervating the anterior knee joint capsule as well as the intraarticular and extraarticular knee ligaments. Initial interest in the genicular nerve included radiofrequency ablation. RFA while effective, also brings with it increased procedure and equipment costs with a non-response rate of around 25%. Alcohol neurolysis of the genicular nerve is being seen as a potential alternative, low-cost, effective option for relieving chronic knee pain in patients. Previous case reports have focused on the use of alcohol, we report the use of phenol for genicular neurolysis. Of the four genicular nerve branches, only three are targeted due to the proximity of the inferolateral genicular nerve to the common peroneal nerve. The purpose of this case report is to show the effectiveness of phenol for neurolysis of the genicular nerves in relieving chronic knee pain in patients in which surgery is not an option.

The Effect of Metformin on Vestibular Schwannoma Growth: A Systematic Review and Meta-analysis.

OBJECTIVES: To systematically review and evaluate metformin's potential impact on vestibular schwannoma (VS) growth. DATA SOURCES: PubMed, Cochrane Library, and Embase. REVIEW METHODS: A retrospective cohort study was performed on sporadic VS patients undergoing initial observation who had at least two magnetic resonance imaging studies. Patients were stratified by metformin use during the observation period. Primary endpoint was VS growth, defined as at least a 2 mm increase in diameter. Survival free of tumor growth was evaluated between groups. Systematic review and meta-analysis were performed to produce a pooled odds ratio [OR]. Study heterogeneity was assessed and post-hoc power analysis was performed. RESULTS: A total of 123 patients were included, of which 17% were taking metformin. Median patient age was 56.6 years (range, 25.1-84.5). There were no statistically significant differences between the groups. Survival analysis did not demonstrate a statistically significant difference in time to VS growth between groups (hazard ratio = 0.61, 95% confidence interval [CI] = 0.29-1.29). Furthermore, logistic regression analysis did not demonstrate a statistically significant difference between groups in the odds of VS growth (OR = 0.46, 95% CI = 0.17-1.27). Systematic review identified 3 studies. Meta-analysis suggested that metformin reduces the odds of developing VS growth (pooled OR = 0.45, 95% CI = 0.29-0.71). Studies demonstrated low between-study heterogeneity. Power analysis demonstrated a sample size of 220 patients with equal randomization would be required to prospectively identify a true difference with 80% power. CONCLUSIONS: Metformin use may reduce the odds of VS growth. A randomized trial would be ideal to identify an unbiased estimate of metformin's effect on VS growth. Laryngoscope, 133:2066-2072, 2023.

Survival Outcomes of Patients with Mycosis Fungoides Involving the External Ear and Ear Canal.

OBJECTIVES/HYPOTHESIS: Mycosis Fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma. Disease involvement of specific locations may be more significant than simply the symptoms associated with that site; it is possible that involvement of certain sites could be associated with poor prognosis. We aimed to evaluate the outcomes of patients with MF with documented involvement of the EAC and external ear. STUDY DESIGN: Retrospective analysis. METHODS: We retrospectively reviewed 40 patients with MF that were treated by otologists between 2012 and 2021. RESULTS: We report the largest series of patients with MF involving the external ear and EAC. Of the 40 patients included in this study, 17 presented with Mycosis Fungoides in the otologic region (MFO). Of these 17 MFO patients, 2/17 had involvement of the external ear only, 3/17 of the EAC only, 11/17 of both the external ear and EAC, and 1/17 of the periauricular skin. Of note, 11/14 (79%) patients presenting with EAC disease died compared to11/26 (42%) of patients without involvement. In addition, eight of the 13 (62%) patients with external ear involvement died compared to 14/27 (52%) of patients without involvement. Ear canal involvement was associated with a statistically significant shorter overall survival duration in patients with MF (p = 0.03). Furthermore, disease in the EAC was found to have a hazard ratio value of 2.565 (CI 1.102-5.970). CONCLUSIONS: Involvement of the EAC by MF portends a poor prognosis. This finding highlights the need for a more in-depth otologic evaluation of patients with MF. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:1486-1491, 2023.