5-Aminolevulinic Acid Fluorescence-Guided Surgery in Head and Neck Squamous Cell Carcinoma.

OBJECTIVES: To determine the utility of 5-aminolevulinic acid (5-ALA) fluorescence for resection of head and neck carcinoma. METHODS: In this prospective pilot trial, 5-ALA was administered as an oral suspension 3-5 h prior to induction of anesthesia for resection of head and neck squamous cell carcinoma (HNSCC). Following resection, 405 nm blue light was applied, and fluorescence of the tumor as well as the surgical bed was recorded. Specimen fluorescence intensity was graded categorically as none (score = 0), mild (1), moderate (2), or robust (3) by the operating surgeon intraoperatively and corroborated with final pathologic diagnosis. RESULTS: Seven patients underwent resection with 5-ALA. Five (83%) were male with an age range of 33-82 years (mean = 60). Sites included nasal cavity (n = 3), oral cavity (n = 3), and the larynx (n = 1). All specimens demonstrated robust fluorescence when 5-ALA was administered 3-5 h preoperatively. 5-ALA fluorescence predicted the presence of perineural invasion, a positive margin, and metastatic lymphadenopathy. Two patients had acute photosensitivity reactions, and one patient had a temporary elevation of hepatic enzymes. CONCLUSIONS: 5-ALA induces robust intraoperative fluorescence of HNSCC, capable of demonstrating a positive margin, perineural invasion, and metastatic nodal disease. Although no conclusions are there about the safety of this drug in the head and neck cancer population, our study parallels the extensive safety data in the neurosurgical literature. Future applications may include intraoperative assessment of margin status, diagnostic accuracy, and impacts on survival. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:741-748, 2024.

Gabapentin and postoperative pain and opioid consumption: A double-blind randomized controlled trial of perioperative pain management for sinus surgery.

BACKGROUND: The link between post-operative narcotic prescription and opioid misuse has spurred a nationwide effort to reduce perioperative opioid use. Previous work has suggested that perioperative gabapentin may reduce post-operative pain and opioid consumption across different procedures, although the optimal regimen remains to be defined. METHODS: Chronic rhinosinusitis (CRS) patients undergoing functional endoscopic sinus surgery (FESS) with or without septoplasty were randomized to receive a 7-day pre- and post-operative course of placebo or gabapentin, starting at 300 mg daily and titrated to 300 mg three times daily, in a double-blind fashion. Primary endpoint was pain level using a validated visual analog scale (VAS). Secondary endpoints included post-operative opioid consumption and side effects, as well as modified Lund-Kennedy endoscopy, Lund-Mackay, and SNOT-22 scores. RESULTS: Analysis of 35 patients (20 gabapentin, 15 control) showed no significant difference in mean postoperative VAS (p = 0.18) or postoperative opioid consumption between the placebo and gabapentin groups (2.3 and 4.8 oxycodone tablets respectively, p = 0.18). 15 of 35 patients did not require any post-operative oxycodone tablets, and only two patients required more than six tablets. CONCLUSION: Preliminary results show no significant change in pain after FESS with or without septoplasty in patients taking 7-day pre- and post-operative gabapentin versus placebo. Results also showed no significant difference in opioid consumption between the treatment and placebo groups. Post-operative pain scores and opioid requirements are both quite low following FESS. Many patients do not need opioids at all, suggesting that routine initial post-operative opioid prescriptions can be limited accordingly.

Cell volume expansion and local contractility drive collective invasion of the basement membrane in breast cancer.

Breast cancer becomes invasive when carcinoma cells invade through the basement membrane (BM)-a nanoporous layer of matrix that physically separates the primary tumour from the stroma. Single cells can invade through nanoporous three-dimensional matrices due to protease-mediated degradation or force-mediated widening of pores via invadopodial protrusions. However, how multiple cells collectively invade through the physiological BM, as they do during breast cancer progression, remains unclear. Here we developed a three-dimensional in vitro model of collective invasion of the BM during breast cancer. We show that cells utilize both proteases and forces-but not invadopodia-to breach the BM. Forces are generated from a combination of global cell volume expansion, which stretches the BM, and local contractile forces that act in the plane of the BM to breach it, allowing invasion. These results uncover a mechanism by which cells collectively interact to overcome a critical barrier to metastasis.

SLC12A9 is a lysosome-detoxifying ammonium - chloride co-transporter.

Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH 4 + ). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal ammonium exporter that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 is a chloride-driven ammonium co-transporter that is central in an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.

Deterministic evolution and stringent selection during preneoplasia.

The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.

Seeking an optimal dose of preoperative corticosteroids in chronic rhinosinusitis with nasal polyposis: A randomized controlled trial.

BACKGROUND: Preoperative corticosteroids have been shown to improve surgical visibility and intraoperative blood loss for chronic rhinosinusitis with nasal polyposis (CRSwNP) patients undergoing endoscopic sinus surgery (ESS). However, there is no consensus on the optimal dosing regimen. METHODS: A randomized, controlled trial was conducted to compare low, medium, and high dose corticosteroids prior to ESS. Patients with CRSwNP refractory to medical management were randomized to low (N = 8), medium (N = 10), or high (N = 5) dosing regimens of corticosteroids prior to ESS. Baseline disease severity was measured with the 22-item Sino-nasal Outcome Test and Lund-Mackay scores. Modified Lund-Kennedy endoscopic scores (MLKES) were measured at baseline and after corticosteroid treatment. Intraoperative parameters were measured including Boezaart surgical visibility score, intraoperative blood loss, and operative time. RESULTS: Medium dose corticosteroids demonstrated a superior surgical visibility score to low dose and comparable results to high dose, but these results were not significant (p = 0.33). No significant difference was observed between groups for total blood loss (p = 0.15), operative time (p = 0.87), or change in MLKES (p = 0.27). CONCLUSIONS: Current recommendations include the use of preoperative corticosteroids in patients with CRSwNP undergoing ESS, but there is no consensus on dose or duration. We did not find a statistically significant difference in surgical field visibility, intraoperative blood loss, or operative time between different dosing regimens. Further studies are needed to evaluate the efficacy of a low-dose preoperative regimen with the goal of reducing cumulative patient exposure to systemic corticosteroids.

Association Between Race and Opioid-Induced Respiratory Depression: An International Post Hoc Analysis of the Prediction of Opioid-induced Respiratory Depression In Patients Monitored by Capnography Trial.

BACKGROUND: Opioid-induced respiratory depression (OIRD) is common on the medical and surgical wards and is associated with increased morbidity and health care costs. While previous studies have investigated risk factors for OIRD, the role of race remains unclear. We aim to investigate the association between race and OIRD occurrence on the medical/surgical ward. METHODS: This is a post hoc analysis of the PRediction of Opioid-induced respiratory Depression In patients monitored by capnoGraphY (PRODIGY) trial; a prospective multinational observational blinded study of 1335 general ward patients who received parenteral opioids and underwent blinded capnography and oximetry monitoring to identify OIRD episodes. For this study, demographic and perioperative data, including race and comorbidities, were analyzed and assessed for potential associations with OIRD. Univariable χ 2 and Mann-Whitney U tests were used. Stepwise selection of all baseline and demographic characteristics was used in the multivariable logistic regression analysis. RESULTS: A total of 1253 patients had sufficient racial data (317 Asian, 158 Black, 736 White, and 42 other races) for inclusion. The incidence of OIRD was 60% in Asians (N = 190/317), 25% in Blacks (N = 40/158), 43% in Whites (N = 316/736), and 45% (N = 19/42) in other races. Baseline characteristics varied significantly: Asians were older, more opioid naïve, and had higher opioid requirements, while Blacks had higher incidences of heart failure, obesity, and smoking. Stepwise multivariable logistic regression revealed that Asians had increased risk of OIRD compared to Blacks (odds ratio [OR], 2.49; 95% confidence interval [CI], 1.54-4.04; P = .0002) and Whites (OR, 1.38; 95% CI, 1.01-1.87; P = .0432). Whites had a higher risk of OIRD compared to Blacks (OR, 1.81; 95% CI, 1.18-2.78; P = .0067). The model's area under the curve was 0.760 (95% CI, 0.733-0.787), with a Hosmer-Lemeshow goodness-of-fit test P value of .23. CONCLUSIONS: This post hoc analysis of PRODIGY found a novel association between Asian race and increased OIRD incidence. Further study is required to elucidate its underlying mechanisms and develop targeted care pathways to reduce OIRD in susceptible populations.

Sinonasal Issues After Maxillectomy With Free Flap Reconstruction: Incidence and Clinical Approach.

OBJECTIVE: To characterize the clinical features associated with sinonasal complaints after maxillectomy with free flap reconstruction as well as propose a screening and treatment algorithm. METHODS: Retrospective review of patients who underwent maxillectomy and free flap reconstruction at a tertiary care center. RESULTS: Fifty-eight patients were included, 25 (43.1%) of them had documented sinonasal complaints postoperatively. Eleven patients subsequently underwent revision surgery for sinonasal complaints. Among the 25 patients with sinonasal complaints, 22 patients (88.0%) had nasal crusting, 17 (68.0%) had nasal obstruction, 12 (48.0%) had rhinorrhea, 9 (36.0%) had facial pain or pressure, and 7 (28.0%) had foul odor. Twenty-two patients (88.0%) had multiple sinonasal complaints. There was a higher incidence of both sinonasal complaints and surgical intervention in patients who underwent adjuvant radiation, but this was not statistically significant (47.7% vs 28.6%, P = .235; 29.4% vs 7.1%, P = .265). CONCLUSIONS: Sinonasal complaints are common following free flap reconstruction for a maxillectomy defect and should be screened for at postoperative visits, with early referral to a rhinologist for consideration of endoscopic sinus surgery. Nonsurgical treatment strategies include large-volume nasal saline irrigations, xylitol irrigations for persistent inflammatory symptoms, and culture-directed antibiotic irrigations for persistent infectious symptoms. LEVEL OF EVIDENCE: 4 Laryngoscope, 132:67-72, 2022.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

Long Term Olfactory Outcomes Following Frontal Sinus Surgery in Chronic Rhinosinusitis.

OBJECTIVES/HYPOTHESIS: No studies have evaluated the impact of the types of frontal sinus surgery (FSS) on objective olfaction scores. This study evaluated olfactory function and quality of life (QOL) in chronic rhinosinusitis (CRS) patients before and after total ethmoidectomy with frontal sinusotomy (FS). STUDY DESIGN: Prospective cohort study. METHODS: A prospective study of adult CRS patients undergoing FSS (Draf 2 or Draf 3 procedures) was conducted at a tertiary care center. Primary outcomes included brief smell identification test (BSIT) and sinonasal outcome test-22 (SNOT-22), which were assessed during preoperative evaluation, 6 to 9 weeks postoperatively, and 12 to 24 weeks postoperatively. Normosmia was defined as BSIT ≥9. Statistical significance was determined using the Wilcoxon signed-rank test with α = .05. RESULTS: Thirty-eight patients followed up 12 to 24 weeks after FSS. The differences between baseline and long-term outcomes for BSIT (6.11 vs. 8.24, P = .00034) and SNOT-22 (55.49 vs. 24.32, P < .00001) scores were found to be statistically significant. Although both subgroups had clinically significant olfactory improvements, only the Draf 2 cohort experienced a statistically significant improvement in olfaction at long-term follow-up. There was no statistically significant change in data from 6 to 9 weeks to 12 to 24 weeks postoperatively. CONCLUSIONS: Patients undergoing total ethmoidectomy with FS demonstrated statistically significant increases in olfaction and QOL at long-term postoperative follow-up. This study demonstrated that FS does not negatively impact the olfactory improvement seen in sinus surgery. The lack of statistically significant changes in these olfactory metrics from short to long-term follow-up suggests that there is no additional negative effect of FSS in the long term. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:2173-2178, 2021.

The value of 68 Gallium-DOTATATE PET/CT in sinonasal neuroendocrine tumor management: A case series.

BACKGROUND: 68 Gallium-DOTATATE (68 Ga-DOTATATE) is a somatostatin analog used as a PET tracer to successfully identify neuroendocrine tumors (NETs). Due to the rarity of sinonasal NETs, there are few recommendations for 68 Ga-DOTATATE imaging in these patients. METHODS: We discussed the impact of 68 Ga-DOTATATE imaging on the management of six sinonasal NET cases and reviewed existing literature. RESULTS: 68 Ga-DOTATATE PET/CT revealed an unknown primary in one case and identified metastatic disease in a primary sinonasal small cell neuroendocrine carcinoma (SNEC) patient missed on conventional imaging. In two esthesioneuroblastoma (ENB) patients, 68 Ga-DOTATATE detected abnormal radiotracer uptake not present on 18F-FDG PET/CT and identified a patient for treatment with 177 Lu-DOTATATE. CONCLUSIONS: This is the one of the first few reports, and the largest series to our knowledge, demonstrating the utility of 68 Ga-DOTATATE imaging for primary sinonasal SNEC and ENB. Further study is required to determine its role in sinonasal NET management.

Enhancing Patient Experience in Office-Based Laryngology Procedures With Passive Virtual Reality.

OBJECTIVES: Virtual reality (VR) has been used as nonpharmacologic anxiolysis benefiting patients undergoing office-based procedures. There is little research on VR use in laryngology. This study aims to determine the efficacy of VR as anxiolysis for patients undergoing in-office laryngotracheal procedures. STUDY DESIGN: Randomized controlled trial. SETTING: Tertiary care center. METHODS: Adult patients undergoing office-based larynx and trachea injections, biopsy, or laser ablation were recruited and randomized to receive standard care with local anesthesia only or local anesthesia with adjunctive VR. Primary end point was procedural anxiety measured by the Subjective Units of Distress Scale (SUDS). Subjective pain, measured using a visual analog scale, satisfaction scores, and procedure time, and baseline anxiety, measured using the Hospital Anxiety and Depression Scale (HADS), were also collected. RESULTS: Eight patients were randomized to the control group and 8 to the VR group. SUDS scores were lower in the VR group than in the control group with mean values of 26.25 and 53.13, respectively (P = .037). Baseline HADS scores did not differ between groups. There were no statistically significant differences in pain, satisfaction, or procedure time. Average satisfaction scores in VR and control groups were 6.44 and 6.25, respectively (P = .770). Average pain scores were 3.53 and 2.64, respectively (P = .434). CONCLUSION: This pilot study suggests that VR distraction may be used as an adjunctive measure to decrease patient anxiety during office-based laryngology procedures. Procedures performed using standard local anesthesia resulted in low pain scores and high satisfaction scores even without adjunctive VR analgesia. LEVEL OF EVIDENCE: 1.

Virtual Reality as Distraction Analgesia and Anxiolysis for Pediatric Otolaryngology Procedures.

OBJECTIVES/HYPOTHESIS: While virtual reality (VR) has been used as analgesia and anxiolysis for invasive procedures, no literature exists on the use of VR in the pediatric otolaryngology setting. The purpose of this study was to determine the efficacy of VR in reducing pain and anxiety for pediatric otolaryngology patients. STUDY DESIGN: Randomized controlled trial. METHODS: A total of 53 patients aged 7-17 undergoing in-office nasal endoscopies were included. Patients were randomized to receive VR or standard of care. Procedural pain, anxiety, and satisfaction scores were recorded from patients and caregivers. The physician filled out a childhood emotional manifestation scale (CEMS). RESULTS: Patients in VR group reported a significant decrease in pain (0.80 ± 1.06 vs. 2.26 ± 2.38, P = .018) and anxiety (9.50 ± 12.48 vs. 38.48 ± 29.83, P = .0002) and increase in procedural satisfaction (6.40 ± 0.77 vs. 4.74 ± 1.74, P = .0002) compared to patients in control group. CEMS scores were significantly reduced in VR group (5.15 ± 0.46 vs. 9.64 ± 5.66, P = .0001) and caregiver anxiety levels were significantly reduced in VR group (11.50 ± 17.67 vs. 27.39 ± 30.48, P = .041) compared to control group. There were no reported side effects. Procedural time did not significantly differ between groups. CONCLUSIONS: For pediatric otolaryngology patients undergoing in-office nasal endoscopies, VR is a safe and effective form of distraction analgesia and anxiolysis, significantly reducing pain and increasing procedural satisfaction for patients. In addition, VR significantly reduces anxiety for both patients and caregivers without disrupting procedural efficiency and workflow. LEVEL OF EVIDENCE: 2. Laryngoscope, 131:E1714-E1721, 2021.

Endoscopic Muscle Repair of Right Internal Carotid Artery Rupture Following Endovascular Procedure.

Carotid artery blowout syndrome (CBS) is a deadly complication usually linked to head and neck cancer therapy. We present a different etiology of endoscopic CBS, a complication of endovascular coiling of an intracranial aneurysm, treated with sternocleidomastoid (SCM) muscle graft packing. Case Presentation: An otherwise healthy 55-year-old female presented to the emergency room with right-sided painless vision loss of 23 days. Computed tomography angiography demonstrated a right ophthalmic ICA aneurysm eroding into the right sphenoid sinus with optic nerve compression. Attempted endovascular repair of the aneurysm was complicated by ICA rupture into the sphenoid. An endovascular balloon was inflated proximal to the aneurysm to reduce hemorrhage as ENT performed an endoscopic sphenoidotomy. A hematoma was seen overlying the aneurysm in the superior lateral sphenoid sinus. Layers of SCM muscle were morselized and packed serially. Post-repair angiography showed no further extravasation. Aggressive antiplatelet therapy was initiated. Packing was removed after 14 days. Twenty days postoperatively, the patient had profuse left-sided epistaxis requiring a left sphenopalatine artery ligation. The patient's vision recovered. Discussion: Whereas CBS is often managed by endovascular coil embolism, in our case CBS was caused by this very treatment itself. This case shows the use of SCM muscle graft as an effective repair modality of ICA rupture due to endovascular coiling. Laryngoscope, 131:E764-E766, 2021.

Letter to the editor: Study Summary - Randomized Control Trial of Omega-3 Fatty Acid Supplementation for the Treatment of COVID-19 Related Olfactory Dysfunction.

OBJECTIVES: To evaluate a therapeutic role for omega-3 fatty acid supplementation in the treatment of olfactory dysfunction associated with COVID-19 infection TRIAL DESIGN: Randomized, double-blinded, placebo-controlled trial PARTICIPANTS: Eligible patients are adults with self-reported new-onset olfactory dysfunction of any duration associated with laboratory-confirmed or clinically suspected COVID-19 patients. Exclusion criteria include patients with pre-existing olfactory dysfunction, history of chronic rhinosinusitis or history of sinus surgery, current use of nasal steroid sprays or omega-3 supplementation, fish allergy, or inability to provide informed consent for any reason. The trial is conducted at Mount Sinai Hospital INTERVENTION AND COMPARATOR: The intervention group will receive 2000 mg daily of omega-3 supplementation in the form of two "Fish Oil, Ultra Omega-3" capsules (product of Pharmavite®) daily. The comparator group will take 2 placebo capsules of identical size, shape, and odor daily for 6 weeks. MAIN OUTCOMES: Each subject will take a Brief Smell Identification Test at study enrolment and completion after 6 weeks. The primary outcome will be change in Brief Smell Identification Test over the 6-week period. RANDOMISATION: Patients will be randomized by the Investigational Drug Pharmacy at the Icahn School of Medicine at Sinai via a computer-generated sequence in a 1:1 allocation to treatment or control arms. BLINDING (MASKING): Both participants and researchers will be blinded. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): There will be 88 participants randomized to each group. A total of 176 participants will be randomized. TRIAL STATUS: Protocol Version 1, 8/3/2020 Recruitment is ongoing, started 8/5/2020 with estimated completion 11/30/2020. TRIAL REGISTRATION: The trial is registered on ClinicalTrials.gov with Protocol Identifier: NCT04495816 . TRIAL REGISTRATION: ClinicalTrials.gov, NCT04495816 . Registered 3 August 2020 FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1).

Secondary Intention Healing After Mohs Surgical Excision as an Alternative to Surgical Repair: Evaluation of Wound Characteristics and Esthetic Outcomes.

BACKGROUND: A multitude of reconstructive options exist for patients after Mohs surgery of cutaneous neoplasms of the head and neck. Secondary intention healing is often overlooked and underused but has numerous advantages, including superior esthetic outcomes compared with surgical reconstruction for wounds that exhibit particular characteristics. The ability to predict cosmetic results based on wound characteristics can greatly help in the decision between surgical repair and secondary intention healing. Although other studies have discussed results after secondary intention healing on various areas of the head and neck, here, we specifically focus on cases of the nasal area. METHODS: We conducted a chart review of 37 patients with nasal reconstructions using secondary intention healing by a single surgeon over a 2-year period. Wound outcomes were graded as poor, acceptable, good, or excellent based on definitions found in the literature. RESULTS: We found that overall, the best cosmetic outcomes were associated with concave areas of the nose, such as the nasal ala and sidewall, and that superficial wounds healed better than deep wounds. Furthermore, we found that convex areas of the nose, such as the nasal tip, did not heal as well by secondary intention. However, if the wound was small and superficial enough, the wound still healed with a good to excellent cosmetic outcome. CONCLUSIONS: Healing by secondary intention is a reasonable consideration for suitable wounds. The need for surgical scar revision is addressed, if necessary, after the wound has healed. The benefits of secondary intention healing include:Future studies will address a larger cohort size of patients with more varied skin types and ages, as these are characteristics that can influence cosmetic outcome. Furthermore, healed wounds continue to improve in appearance over time, and it would be worthwhile to monitor patients' cosmetic outcomes over a longer follow-up period.

The RUNX1-ETO target gene RASSF2 suppresses t(8;21) AML development and regulates Rac GTPase signaling.

Large-scale chromosomal translocations are frequent oncogenic drivers in acute myeloid leukemia (AML). These translocations often occur in critical transcriptional/epigenetic regulators and contribute to malignant cell growth through alteration of normal gene expression. Despite this knowledge, the specific gene expression alterations that contribute to the development of leukemia remain incompletely understood. Here, through characterization of transcriptional regulation by the RUNX1-ETO fusion protein, we have identified Ras-association domain family member 2 (RASSF2) as a critical gene that is aberrantly transcriptionally repressed in t(8;21)-associated AML. Re-expression of RASSF2 specifically inhibits t(8;21) AML development in multiple models. Through biochemical and functional studies, we demonstrate RASSF2-mediated functions to be dependent on interaction with Hippo kinases, MST1 and MST2, but independent of canonical Hippo pathway signaling. Using proximity-based biotin labeling we define the RASSF2-proximal proteome in leukemia cells and reveal association with Rac GTPase-related proteins, including an interaction with the guanine nucleotide exchange factor, DOCK2. Importantly, RASSF2 knockdown impairs Rac GTPase activation, and RASSF2 expression is broadly correlated with Rac-mediated signal transduction in AML patients. Together, these data reveal a previously unappreciated mechanistic link between RASSF2, Hippo kinases, and Rac activity with potentially broad functional consequences in leukemia.

Hippo kinase loss contributes to del(20q) hematologic malignancies through chronic innate immune activation.

Heterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression, we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these 2 molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1ß and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1ß production in the JAK2-V617F MPN model. This study identified Hippo kinase MST1 (STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and revealed a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.

Differential microglia and macrophage profiles in human IDH-mutant and -wild type glioblastoma.

Microglia and macrophages are the largest component of the inflammatory infiltrate in glioblastoma (GBM). However, whether there are differences in their representation and activity in the prognostically-favorable isocitrate dehydrogenase (IDH)-mutated compared to -wild type GBMs is unknown. Studies on human specimens of untreated IDH-mutant GBMs are rare given they comprise 10% of all GBMs and often present at lower grades, receiving treatments prior to dedifferentiation that can drastically alter microglia and macrophage phenotypes. We were able to obtain large samples of four previously untreated IDH-mutant GBM. Using flow cytometry, immunofluorescence techniques with automated segmentation protocols that quantify at the individual-cell level, and comparison between single-cell RNA-sequencing (scRNA-seq) databases of human GBM, we discerned dissimilarities between GBM-associated microglia and macrophages (GAMMs) in IDH-mutant and -wild type GBMs. We found there are significantly fewer GAMM in IDH-mutant GBMs, but they are more pro-inflammatory, suggesting this contributes to the better prognosis of these tumors. Our pro-inflammatory score which combines the expression of inflammatory markers (CD68/HLA-A, -B, -C/TNF/CD163/IL10/TGFB2), Iba1 intensity, and GAMM surface area also indicates that more pro-inflammatory GAMMs are associated with longer overall survival independent of IDH status. Interrogation of scRNA-seq databases demonstrates microglia in IDH-mutants are mainly pro-inflammatory, while anti-inflammatory macrophages that upregulate genes such as FCER1G and TYROBP predominate in IDH-wild type GBM. Taken together, these observations are the first head-to-head comparison of GAMMs in treatment-naïve IDH-mutant versus -wild type GBMs. Our findings highlight biological disparities in the innate immune microenvironment related to IDH prognosis that can be exploited for therapeutic purposes.

Rethinking Causation in Cancer with Evolutionary Developmental Biology.

Despite the productivity of basic cancer research, cancer continues to be a health burden to society because this research has not yielded corresponding clinical applications. Many proposed solutions to this dilemma have revolved around implementing organizational and policy changes related to cancer research. Here I argue for a different solution: a new conceptualization of causation in cancer. Neither the standard molecular biomarker approaches nor evolutionary biology approaches to cancer fully capture its complex causal dynamics, even when considered jointly. These approaches map on to Ernst Mayr's proximate-ultimate distinction, which is an inadequate conceptualization of causation in biological systems and makes it difficult to connect developmental and evolutionary viewpoints. I propose looking to evolutionary developmental biology (EvoDevo) to overcome the distinction and integrate the proximate and ultimate causal frameworks. I use the concepts of modularity and evolvability to show how an EvoDevo perspective can be manifested in cancer translational research. This perspective on causation in cancer is better suited for integrating the complexity of current empirical results and can facilitate novel developments in the investigation and clinical treatment of cancer.