Loss of CREBBP and KMT2D cooperate to accelerate lymphomagenesis and shape the lymphoma immune microenvironment.

Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8+ T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.

Genome and clonal hematopoiesis stability contrasts with immune, cfDNA, mitochondrial, and telomere length changes during short duration spaceflight.

Background: The Inspiration4 (I4) mission, the first all-civilian orbital flight mission, investigated the physiological effects of short-duration spaceflight through a multi-omic approach. Despite advances, there remains much to learn about human adaptation to spaceflight's unique challenges, including microgravity, immune system perturbations, and radiation exposure. Methods: To provide a detailed genetics analysis of the mission, we collected dried blood spots pre-, during, and post-flight for DNA extraction. Telomere length was measured by quantitative PCR, while whole genome and cfDNA sequencing provided insight into genomic stability and immune adaptations. A robust bioinformatic pipeline was used for data analysis, including variant calling to assess mutational burden. Result: Telomere elongation occurred during spaceflight and shortened after return to Earth. Cell-free DNA analysis revealed increased immune cell signatures post-flight. No significant clonal hematopoiesis of indeterminate potential (CHIP) or whole-genome instability was observed. The long-term gene expression changes across immune cells suggested cellular adaptations to the space environment persisting months post-flight. Conclusion: Our findings provide valuable insights into the physiological consequences of short-duration spaceflight, with telomere dynamics and immune cell gene expression adapting to spaceflight and persisting after return to Earth. CHIP sequencing data will serve as a reference point for studying the early development of CHIP in astronauts, an understudied phenomenon as previous studies have focused on career astronauts. This study will serve as a reference point for future commercial and non-commercial spaceflight, low Earth orbit (LEO) missions, and deep-space exploration.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

ChromaFold predicts the 3D contact map from single-cell chromatin accessibility.

The identification of cell-type-specific 3D chromatin interactions between regulatory elements can help to decipher gene regulation and to interpret the function of disease-associated non-coding variants. However, current chromosome conformation capture (3C) technologies are unable to resolve interactions at this resolution when only small numbers of cells are available as input. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps and regulatory interactions from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility profiles across metacells, and predicted CTCF motif tracks as input features and employs a lightweight architecture to enable training on standard GPUs. Once trained on paired scATAC-seq and Hi-C data in human cell lines and tissues, ChromaFold can accurately predict both the 3D contact map and peak-level interactions across diverse human and mouse test cell types. In benchmarking against a recent deep learning method that uses bulk ATAC-seq, DNA sequence, and CTCF ChIP-seq to make cell-type-specific predictions, ChromaFold yields superior prediction performance when including CTCF ChIP-seq data as an input and comparable performance without. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations. ChromaFold thus achieves state-of-the-art prediction of 3D contact maps and regulatory interactions using scATAC-seq alone as input data, enabling accurate inference of cell-type-specific interactions in settings where 3C-based assays are infeasible.

Canadian multidisciplinary expert consensus on the use of biologics in upper airways: a Delphi study.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) often coexists with lower airway disease. With the overlap between upper and lower airway disease, optimal management of the upper airways is undertaken in conjunction with that of the lower airways. Biologic therapy with targeted activity within the Type 2 inflammatory pathway can improve the clinical signs and symptoms of both upper and lower airway diseases. Knowledge gaps nevertheless exist in how best to approach patient care as a whole. There have been sixteen randomized, double-blind, placebo-controlled trails performed for CRSwNP targeted components of the Type 2 inflammatory pathway, notably interleukin (IL)-4, IL-5 and IL-13, IL- 5R, IL-33, and immunoglobulin (Ig)E. This white paper considers the perspectives of experts in various disciplines such as rhinology, allergy, and respirology across Canada, all of whom have unique and valuable insights to contribute on how to best approach patients with upper airway disease from a multidisciplinary perspective. METHODS: A Delphi Method process was utilized involving three rounds of questionnaires in which the first two were completed individually online and the third was discussed on a virtual platform with all the panelists. A national multidisciplinary expert panel of 34 certified specialists was created, composed of 16 rhinologists, 7 allergists, and 11 respirologists who evaluated the 20 original statements on a scale of 1-9 and provided comments. All ratings were quantitively reviewed by mean, median, mode, range, standard deviation and inter-rater reliability. Consensus was defined by relative interrater reliability measures-kappa coefficient ([Formula: see text]) value > 0.61. RESULTS: After three rounds, a total of 22 statements achieved consensus. This white paper only contains the final agreed upon statements and clear rationale and support for the statements regarding the use of biologics in patients with upper airway disease. CONCLUSION: This white paper provides guidance to Canadian physicians on the use of biologic therapy for the management of upper airway disease from a multidisciplinary perspective, but the medical and surgical regimen should ultimately be individualized to the patient. As more biologics become available and additional trials are published we will provide updated versions of this white paper every few years.

Cooperative super-enhancer inactivation caused by heterozygous loss of CREBBP and KMT2D skews B cell fate decisions and yields T cell-depleted lymphomas.

Mutations affecting enhancer chromatin regulators CREBBP and KMT2D are highly co-occurrent in germinal center (GC)-derived lymphomas and other tumors, even though regulating similar pathways. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) indeed accelerated lymphomagenesis. C+K haploinsufficiency induced GC hyperplasia by altering cell fate decisions, skewing B cells away from memory and plasma cell differentiation. C+K deficiency particularly impaired enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for immunoregulatory and differentiation genes. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other's stable recruitment to enhancers. Notably, C+K lymphomas in mice and humans manifested significantly reduced CD8 + T-cell abundance. Hence, deficiency of C+K cooperatively induced an immune evasive phenotype due at least in part to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions. SIGNIFICANCE: Although CREBBP and KMT2D have similar enhancer regulatory functions, they are paradoxically co-mutated in lymphomas. We show that their combined loss causes specific disruption of super-enhancers driving immune synapse genes. Importantly, this leads to reduction of CD8 cells in lymphomas, linking super-enhancer function to immune surveillance, with implications for immunotherapy resistance.

BTG1 mutation yields supercompetitive B cells primed for malignant transformation.

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Structure-activity relationship studies of benzothiazole-phenyl analogs as multi-target ligands to alleviate pain without affecting normal behavior.

Soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) are potential targets for several diseases. Previous studies have reported that concomitant selective inhibition of sEH and FAAH produced antinociception effects in an animal model of pain. However, the co-administration of a selective sEH inhibitor and a selective FAAH inhibitor might produce serious side effects due to drug-drug interactions that could complicate drug development in the long term. Thus, discovering dual sEH/FAAH inhibitors, single small molecules that can simultaneously inhibit both sEH and FAAH, would be a significant accomplishment in the medicinal chemistry field. Herein, we report the synthesis and biological evaluation of benzothiazole-phenyl-based analogs as potential dual sEH/FAAH inhibitors. This work represents a follow-up structure-activity relationship (SAR) and metabolic-stability studies of our best dual sEH/FAAH inhibitor identified previously, as well as in vivo evaluation of its effects on voluntary locomotor behavior in rats. Our SAR study indicates that trifluoromethyl groups on the aromatic rings are well tolerated by the targeted enzymes when placed at the ortho and para positions; however, they, surprisingly, did not improve metabolic stability in liver microsomes. Our behavioral studies indicate that doses of dual sEH/FAAH inhibitors that alleviate pain do not depress voluntary behavior in naïve rats, which is a common side effect of currently available analgesic drugs (e.g., opioids). Thus, dual sEH/FAAH inhibitors may be a safe and effective approach to treat pain.

Seasonal Variation of Peritonsillar Abscess Presentation to an Emergency Department in an Atlantic Canadian Climate: A Retrospective Review.

OBJECTIVES: Patients with a peritonsillar abscess (PTA) often present to emergency departments as the first point of medical contact. Upper respiratory tract infections (URTIs) are more frequent in the winter. Therefore, we hypothesize that the incidence of PTAs will be more frequent in colder winter months as well. This is the first study assessing the seasonal variation and epidemiology of PTA presentations to an emergency department in Atlantic Canada, home to a unique maritime climate. METHODS: A retrospective cohort study was conducted through a chart review of all patients who presented to the Saint John Regional Hospital Emergency Department from January 1, 2015, to December 31, 2020. Patient characteristics, treatment, and microbiology were reported. A chi-square goodness-of-fit test assessed the seasonal variation of PTA. Pearson correlations assessed PTA incidence per mean monthly temperature and humidity. RESULTS: A total of 75 patients were included. 57.3% were male and 42.7% were female, with a mean age (±SD) of 35.9 ± 14.0. Most patients presented afebrile (82.7%, cutoff ≥ 38.0°C). Approximately half of all patients had an elevated WBC count (49.3%, cutoff ≥ 10.9 × 109). The most common bacteria isolated were Streptococcus species followed by anaerobic bacteria (17.9%). No significant variation was found with respect to season (X2(3) = 1.0, P = .801), temperature (r(70) = 0.198, P = .096), or humidity, (r(70) = 0.063, P = .599). CONCLUSION: This study did not find a seasonal variation of PTA in a maritime climate. These findings question the anecdotal hypothesis that PTA is associated with progression from acute URTIs and therefore would be more common in the winter months.

An Aged/Autoimmune B-cell Program Defines the Early Transformation of Extranodal Lymphomas.

A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients. SIGNIFICANCE: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8-11). This article is highlighted in the In This Issue feature, p. 1.

Social Media Presence Across U.S. Neurosurgical Residency Programs and Subspecialties.

OBJECTIVE: U.S. neurosurgery programs are increasingly using social media accounts. We performed a search and analysis of social media accounts across all U.S. neurosurgical training programs with an attempt at understanding the relative utilization by various subspecialties. METHODS: We compiled a list of all Accreditation Council for Graduate Medical Education-accredited U.S. neurosurgery programs and the faculty. Each faculty member was classified on the basis of their subspecialty. Next, the Twitter, Facebook, and Instagram profiles were extensively searched for the number of followers and posts. RESULTS: We analyzed 110 programs with 1829 clinical faculty. Programs with a larger number of faculty (P = 0.035; χ2 = 13.528) and residents (P = 0.003; χ2 = 11.865) were more likely to have a social media account. Likewise, faculty and resident numbers had a positive correlation to Twitter (P = 0.037 for faculty size; P = 0.008 for residents' size) and Instagram followers (P = 0.003 for faculty size; P < 0.001 for residents' size). We additionally found a significant association between subspecialty type and the presence of a Twitter and Instagram account (P = 0.001; P = 0.028) and the number of followers (P = 0.004; P = 0.013), especially the vascular and oncology subspecialties. CONCLUSIONS: Many U.S. neurosurgical programs have social media accounts with larger programs likely to have social media accounts. While there is a larger percentage of spine faculty within individual departments, vascular and oncology subspecialties are more likely to have a Twitter account. We suggest the need for increased engagement among spine faculty across social media platforms.

Teaching cancer imaging in the era of precision medicine: Looking at the big picture.

The role of imaging in cancer diagnosis and treatment has evolved at the same rapid pace as cancer management. Over the last twenty years, with the advancement of technology, oncology has become a multidisciplinary field that allows for researchers and clinicians not only to create individualized treatment options for cancer patients, but also to evaluate patients' response to therapy with increasing precision. Familiarity with these concepts is a requisite for current and future radiologists, as cancer imaging studies represent a significant and growing component of any radiology practice, from tertiary cancer centers to community hospitals. In this review we provide the framework to teach cancer imaging in the era of genomic oncology. After reading this article, readers should be able to illustrate the basics cancer genomics, modern cancer genomics, to summarize the types of systemic oncologic therapies available, their patterns of response and their adverse events, to discuss the role of imaging in oncologic clinical trials and the role of tumor response criteria and to display the future directions of oncologic imaging.

Through the Looking Glass: Unraveling the Stage-Shift of Acute Rejection in Renal Allografts.

Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore the need for more accurate and reflexive immunosurveillance to uncover the flux in alloimmunity between allograft health and the onset and progression of rejection. QSant-a urine based multi-analyte diagnostic test-was developed to profile renal transplant health and prognosticate injury, risk of evolution, and resolution of acute rejection. Q-Score-the composite score, across measurements of DNA, protein and metabolic biomarkers in the QSant assay-enables this risk prognostication. The domain of immune quiescence-below a Q-Score threshold of 32-is well established, based on published AUC of 98% for QSant. However, the trajectory of rejection is variable, given that causality is multi-factorial. Injury and subtypes of rejection are captured by the progression of Q-Score. This publication explores the clinical utility of QSant across the alloimmunity gradient of 32-100 for the early diagnosis of allograft injury and rejection.

Rate of malignancy for thyroid nodules with AUS/FLUS cytopathology in a tertiary care center - a retrospective cohort study.

BACKGROUND: Thyroid nodules are stratified through fine-needle aspiration (FNA) and are often categorized using The Bethesda System for Reporting Thyroid Cytopathology, which estimates the risk of malignancy for six cytopathological categories. The atypia of undetermined significance (AUS) and follicular lesion of undetermined significance (FLUS) categories have varying malignancy rates reported in the literature which can range from 6 to 72.9%. Due to this heterogeneity, we assessed the malignancy rate and effectiveness of repeat FNA (rFNA) for AUS/FLUS thyroid cytopathology at our institution. METHODS: Electronic health records of patients with AUS/FLUS thyroid cytopathology on FNA at our center since the implementation of the Bethesda System on May 1, 2014-December 31, 2019 were retrospectively reviewed. Patient demographics, treatment pathway, and pathology results were collected. The treatment pathway of the nodules, the rFNA results, and the malignant histopathology results were reported. Malignancy rates were calculated as an upper and lower limit estimate. RESULTS: This study described 182 AUS/FLUS thyroid nodules from 177 patients. In total, 24 thyroid nodules were deemed malignant upon histopathology, yielding a final malignancy rate of 13.2-25.3%. All of the malignancies were variants of papillary thyroid carcinoma. The malignancy rate of the nodules which underwent resection without rFNA (21.5%) was lower than the malignancy rate of the nodules which underwent resection after rFNA (43.8%). 45.5% of the rFNA results were re-classified into more definitive categories. CONCLUSION: The malignancy rate of AUS/FLUS thyroid cytopathology at our center is in line with the risk of malignancy stated by the 2017 Bethesda System. However, our malignancy rate is lower than some other Canadian centers and approximately half of our rFNAs were re-classified, highlighting the importance of establishing center-specific malignancy and rFNA re-classification rates to guide treatment decisions.

Choosing Wisely Canada - pediatric otolaryngology recommendations.

The Choosing Wisely Canada campaign raises awareness amongst physicians and patients regarding unnecessary or inappropriate tests and treatments. Using an online survey, members of the Pediatric Otolaryngology Subspecialty Group within the Canadian Society of Otolaryngology - Head & Neck Surgery developed a list of nine evidence based recommendations to help physicians and patients make treatment decisions regarding common pediatric otolaryngology presentations: (1) Don't routinely order a plain film x-ray in the evaluation of nasal fractures; (2) Don't order imaging to distinguish acute bacterial sinusitis from an upper respiratory infection; (3) Don't place tympanostomy tubes in most children for a single episode of otitis media with effusion of less than 3 months duration; (4) Don't routinely prescribe intranasal/systemic steroids, antihistamines or decongestants for children with uncomplicated otitis media with effusion; (5) Don't prescribe oral antibiotics for children with uncomplicated tympanostomy tube otorrhea or uncomplicated acute otitis externa; (6) Don't prescribe codeine for post-tonsillectomy/adenoidectomy pain relief in children; (7) Don't administer perioperative antibiotics for elective tonsillectomy in children; (8) Don't perform tonsillectomy for children with uncomplicated recurrent throat infections if there have been fewer than 7 episodes in the past year, 5 episodes in each of the past 2 years, or 3 episodes in each of the last 3 years; and (9) Don't perform endoscopic sinus surgery for uncomplicated pediatric chronic rhinosinusitis prior to failure of maximal medical therapy and adenoidectomy.

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice.

Existing preclinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1-2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.