MLL-AF4 cooperates with PAF1 and FACT to drive high-density enhancer interactions in leukemia.

Aberrant enhancer activation is a key mechanism driving oncogene expression in many cancers. While much is known about the regulation of larger chromosome domains in eukaryotes, the details of enhancer-promoter interactions remain poorly understood. Recent work suggests co-activators like BRD4 and Mediator have little impact on enhancer-promoter interactions. In leukemias controlled by the MLL-AF4 fusion protein, we use the ultra-high resolution technique Micro-Capture-C (MCC) to show that MLL-AF4 binding promotes broad, high-density regions of enhancer-promoter interactions at a subset of key targets. These enhancers are enriched for transcription elongation factors like PAF1C and FACT, and the loss of these factors abolishes enhancer-promoter contact. This work not only provides an additional model for how MLL-AF4 is able to drive high levels of transcription at key genes in leukemia but also suggests a more general model linking enhancer-promoter crosstalk and transcription elongation.

Unique immune and inflammatory cytokine profiles may define long COVID syndrome.

PURPOSE: Long COVID is estimated to occur in 5-10% of individuals after acute SARS-CoV-2 infection. However, the pathophysiology driving the disease process is poorly understood. METHODS: We evaluated urine and plasma inflammatory and immune cytokine profiles in 33 individuals with long COVID compared to 33 who were asymptomatic and recovered, and 34 without prior infection. RESULTS: Mean urinary leukotriene E4 was significantly elevated among individuals with long COVID compared to asymptomatic and recovered individuals (mean difference 774.2 pg/mL; SD 335.7) and individuals without prior SARS-CoV-2 infection (mean difference 503.1 pg/ml; SD 467.7). Plasma chemokine ligand 6 levels were elevated among individuals with long COVID compared to individuals with no prior SARS-CoV-2 infection (mean difference 0.59 units; SD 0.42). We found no significant difference in angiotensin-converting enzyme 2 antibody levels. Plasma tumor necrosis factor receptor-associated factor 2 (TRAF2) levels were reduced among individuals with long COVID compared to individuals who were asymptomatic and recovered (mean difference = 0.6 units, SD 0.46). Similarly, the mean level of Sarcoma Homology 2-B adapter protein 3 was 3.3 units (SD 1.24) among individuals with long COVID, lower than 4.2 units (SD 1.1) among individuals with recovered, asymptomatic COVID. CONCLUSION: Our findings suggest that further studies should be conducted to evaluate the role of leukotriene E4 as a potential biomarker for a diagnostic test. Furthermore, based on reductions in TRAF2, long COVID may be driven in part by impaired TRAF2-dependent immune-mediated inflammation and potentially immune exhaustion.

Direct correction of haemoglobin E ß-thalassaemia using base editors.

Haemoglobin E (HbE) ß-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE ß-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe ß-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (ß-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations.

Determining chromatin architecture with Micro Capture-C.

Micro Capture-C (MCC) is a chromatin conformation capture (3C) method for visualizing reproducible three-dimensional contacts of specified regions of the genome at base pair resolution. These methods are an established family of techniques that use proximity ligation to assay the topology of chromatin. MCC can generate data at substantially higher resolution than previous techniques through multiple refinements of the 3C method. Using a sequence agnostic nuclease, the maintenance of cellular integrity and full sequencing of the ligation junctions, MCC achieves subnucleosomal levels of resolution, which can be used to reveal transcription factor binding sites analogous to DNAse I footprinting. Gene dense regions, close-range enhancer-promoter contacts, individual enhancers within super-enhancers and multiple other types of loci or regulatory regions that were previously challenging to assay with conventional 3C techniques, are readily observed using MCC. MCC requires training in common molecular biology techniques and bioinformatics to perform the experiment and analyze the data. The protocol can be expected to be completed in a 3 week timeframe for experienced molecular biologists.

Whole-genome sequencing of chronic lymphocytic leukemia identifies subgroups with distinct biological and clinical features.

The value of genome-wide over targeted driver analyses for predicting clinical outcomes of cancer patients is debated. Here, we report the whole-genome sequencing of 485 chronic lymphocytic leukemia patients enrolled in clinical trials as part of the United Kingdom's 100,000 Genomes Project. We identify an extended catalog of recurrent coding and noncoding genetic mutations that represents a source for future studies and provide the most complete high-resolution map of structural variants, copy number changes and global genome features including telomere length, mutational signatures and genomic complexity. We demonstrate the relationship of these features with clinical outcome and show that integration of 186 distinct recurrent genomic alterations defines five genomic subgroups that associate with response to therapy, refining conventional outcome prediction. While requiring independent validation, our findings highlight the potential of whole-genome sequencing to inform future risk stratification in chronic lymphocytic leukemia.

DAT negative warm autoimmune haemolytic anaemia secondary to a mature cystic teratoma refractory to immunosuppressive therapy.

Mature cystic teratomas (MCTs) have a rare but recognised association with IgG-mediated autoimmune haemolytic anaemia (AIHA). We present the first case of an MCT associated with IgA-mediated AIHA. We discuss the diagnostic challenges of patients presenting with a direct antiglobulin test negative AIHA picture and the importance of reviewing these cases with the local transfusion laboratory. We also review the treatment options for MCT associated AIHA. In particular, we note they are typically resistant to immunosuppression and that surgical resection should be considered at an early stage. Although MCTs are a rare cause of AIHA, they can be treated easily and should be excluded in young women presenting with AIHA, especially if resistant to corticosteroids.

SARS-CoV-2 Specific IgG Antibodies Persist Over a 12-Month Period in Oral Mucosal Fluid Collected From Previously Infected Individuals.

Background: Developing an understanding of the antibody response, seroprevalence, and seroconversion from natural infection and vaccination against SARS-CoV-2 will give way to a critical epidemiological tool to predict reinfection rates, identify vulnerable communities, and manage future viral outbreaks. To monitor the antibody response on a larger scale, we need an inexpensive, less invasive, and high throughput method. Methods: Here we investigate the use of oral mucosal fluids from individuals recovered from SARS-CoV-2 infection to monitor antibody response and persistence over a 12-month period. For this cohort study, enzyme-linked immunosorbent assays (ELISAs) were used to quantify anti-Spike(S) protein IgG antibodies in participants who had prior SARS-CoV-2 infection and regularly (every 2-4 weeks) provided both serum and oral fluid mucosal fluid samples for longitudinal antibody titer analysis. Results: In our study cohort (n=42) with 17 males and 25 females with an average age of 45.6 +/- 19.3 years, we observed no significant change in oral mucosal fluid IgG levels across the time course of antibody monitoring. In oral mucosal fluids, all the participants who initially had detectable antibodies continued to have detectable antibodies throughout the study. Conclusions: Based on the results presented here, we have shown that oral mucosal fluid-based assays are an effective, less invasive tool for monitoring seroprevalence and seroconversion, which offers an alternative to serum-based assays for understanding the protective ability conferred by the adaptive immune response from viral infection and vaccination against future reinfections.

Detection of persistent SARS-CoV-2 IgG antibodies in oral mucosal fluid and upper respiratory tract specimens following COVID-19 mRNA vaccination.

COVID-19 mRNA vaccines are highly effective at preventing COVID-19. Prior studies have found detectable SARS-CoV-2 IgG antibodies in oral mucosal specimens of participants with history of COVID-19. To assess the development of oral SARS-CoV-2 IgG antibodies among people who received either the Moderna or Pfizer/BioNTech COVID-19 vaccination series, we developed a novel SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) to quantify the concentrations of oral and nasal mucosal SARS-CoV-2 IgG levels. We enrolled 52 participants who received the Moderna vaccine and 80 participants who received the Pfizer/BioNTech vaccine. Oral mucosal specimens were self-collected by participants prior to or on the day of vaccination, and on days 5, 10, 15, and 20 following each vaccination dose and 30, 60, and 90 days following the second vaccination dose. A subset of the cohort provided additional nasal mucosal specimens at every time point. All participants developed detectable oral mucosal SARS-CoV-2 IgG antibodies by 15 days after the first vaccination dose. There were no significant differences in oral mucosal antibody concentrations once participants were fully vaccinated in the Moderna and Pfizer/BioNTech vaccines. Oral or nasal mucosal antibody testing could be an inexpensive and less invasive alternative to serum antibody testing. Further research is needed to understand the duration of detectable oral or nasal mucosal antibodies and how antibody concentrations change with time.

Treatment patterns and outcomes of unfit and elderly patients with Mantle cell lymphoma unfit for standard immunochemotherapy: A UK and Ireland analysis.

Mantle cell lymphoma (MCL) presenting in elderly, unfit patients represents a clinical challenge. Front-line 'attenuated' or low-intensity immunochemotherapy is often employed, although outcomes are relatively unexplored. We report outcomes of attenuated immunochemotherapy in 95 patients with MCL across 19 centres in the UK and Ireland considered unfit for full-dose rituximab-bendamustine or rituximab-cyclophosphamide, doxorubicin, vincristine, prednisolone (R-CHOP). Regimens examined were rituximab-cyclophosphamide, vincristine, prednisolone (R-CVP) (n = 19), dose-attenuated R-CHOP (n = 22), dose attenuated rituximab-bendamustine (n = 24) and rituximab-chlorambucil (n = 30). The primary outcome was progression-free survival (PFS). The secondary outcomes included overall response, overall survival (OS) and toxicity. The median (range) age was 79 (58-89) years and 50% were aged ≥80 years. The median (range) Cumulative Illness Rating Scale-Geriatric score was 6 (0-24). The median PFS for all patients was 15 months [95% confidence interval (CI) 8·7-21·2) and median OS was 31·4 months (95% CI 19·7-43·2). By multivariable analysis (MVA), the only clinical factor associated with an inferior PFS was blastoid morphology [hazard ratio (HR) 2·90, P = 0·01). Notably, higher treatment intensity (R-CHOP/R-bendamustine composite) provided an independently superior PFS compared with R-CVP/R-chlorambucil (MVA HR 0·49, P = 0·02). Factors associated with inferior OS by MVA were Eastern Cooperative Oncology Group Performance Status (HR 2·14, P = 0·04), blastoid morphology (HR 4·08, P = 0·001) and progression of disease at <24 months status (HR 5·68, P < 0·001). Overall, survival after front-line dose-attenuated immunochemotherapy is unsatisfactory. Clinical trials investigating novel agents such as Bruton tyrosine kinase and B-cell lymphoma 2 inhibitors in this specific clinical setting are warranted.

A level-headed approach to measuring direct oral anticoagulants: A 2-year retrospective analysis of DOAC levels from a tertiary UK centre.

INTRODUCTION: Direct oral anticoagulants (DOAC) are commonly prescribed and measuring drug levels may be useful in a number of contexts. However, data on DOAC level measurement and their clinical utility in real-world studies are limited. METHODS: We carried out a 2-year retrospective cohort study of DOAC levels measured at our institution. RESULTS: One hundred and sixty-nine levels measured in 113 patients were included in the final analysis. Our patients had a median age of 69.9. AF was the commonest indication for anticoagulation. Median turnaround time for inpatient levels was 92 minutes. Median FXa inhibitor levels within 6 hours of last dose and following one half-life were similar to those described previously. However, the range of levels was wider than expected. Importantly, some levels remained in an on therapy range even after 3 half-lives. There was no correlation between dabigatran level and time from last dose. The reason for request varied with setting; 23 outpatient levels were to monitor drug efficacy, whereas 54 and 43 inpatient levels were collected in the context of bleeding and emergency surgery respectively. 60.3% of levels had an impact on clinical decision making. CONCLUSION: Our real-world study demonstrates that DOAC levels can be performed in a timely manner to influence clinical decision making. In addition, it suggests there is a wide variation in levels such that it can be difficult to predict in the real world. Overall, this supports the wider use of DOAC levels to help guide clinicians in managing patients taking these drugs.

An airway traffic jam: a plastic traffic cone masquerading as bronchial carcinoma.

Tracheobronchial foreign body (TFB) aspiration is a common occurrence in children compared with adults. Long-standing cases of TFB aspiration during childhood presenting in an adult have rarely been reported. We report the unique case of an endobronchial Playmobil traffic cone that went undetected for 40 years and presented as a suspected bronchogenic carcinoma. This was subsequently removed successfully with flexible bronchoscopy. To our knowledge this is the first case of a TFB that was overlooked this length of time.

Rifampicin-induced adrenal crisis in a patient with tuberculosis: a therapeutic challenge.

A 55-year-old Indian man presented with productive cough and a large left pleural effusion. Pleural fluid culture grew Mycobacterium tuberculosis, and he was started on antituberculosis therapy. One week later, the patient presented to hospital with drowsiness, dehydration and hypotension. He was transferred to critical care and only improved after starting hydrocortisone and stopping rifampicin. His short synACTHen test subsequently confirmed primary adrenal insufficiency, and a CT of the abdomen showed bilateral adrenal enlargement. Rifampicin is known to accelerate cortisol metabolism. We report the rare case of a rifampicin-induced adrenal crisis as a first presentation of Addison's disease in a patient with tuberculous infiltration of the adrenal glands.

Interscalene brachial plexus block: can the risk of entering the spinal canal be reduced? A study of needle angles in volunteers undergoing magnetic resonance imaging.

BACKGROUND: Spinal cord damage during interscalene brachial plexus block has been attributed to needle entry into the spinal canal. The purpose of this study was to identify the angles and depths of needle insertion that increase the likelihood of such an event, using the traditional classic interscalene approach and two more proximal entry points. METHOD: Magnetic resonance images of the neck from 10 healthy volunteers were used to obtain the three-dimensional spatial coordinates of three skin markers and the right-sided cervical nerves at the exiting neural foramina. The distance of the intervertebral foramina from the skin markers and the angles of the needle vector and the foramina were calculated. RESULTS: The distance from the skin to the intervertebral foramen may be as short as 2.5 cm with the classic approach. A caudal angulation greater than 50 degrees seemed to eliminate the risk of needle entry through the foramen. CONCLUSION: With the classic approach to the interscalene block, there is a greater possibility of the needle passing through the intervertebral foramen if the needle is advanced too deeply. More proximal entry points and techniques that use a more steeply angled needle may reduce the risk of entry into the spinal space.

A study of the characteristics of single-injection insulated block needles in a biologic model.

BACKGROUND AND OBJECTIVES: Single-injection block needles are manufactured in many different lengths, diameters, and tip designs, but the literature contains no reports of methods to assess clinical characteristics of regional-block needles. A novel animal model for the assessment of the characteristics of single-injection regional anesthesia needles is described. METHODS: Nine different needles designed for peripheral nerve blocks that were fitted with identical hubs were used. Pork bellies were used as the biologic model. The bellies were mounted such that the needles passed from inside to outside. The last layer to be penetrated was the skin. Ten experienced and blinded anesthesiologists scored the feel, resistance, and usability of the 9 needles during their passage through similarly prepared pork bellies. Two identical (index) needles were included in the study to assess the internal validity of the study. RESULTS: The overall scoring was acceptably consistent and repeatable and showed statistically significant differences between the needles tested. The needles that were judged the most usable were those with a moderate resistance to passage through the tissue and a high degree of feel, which was defined as the ability to appreciate the passage of the needle through the tissue planes. Needles with very high or very low resistances and those with poor feel scored poorly on the usability scale. Differences in individuals' assessment of the index needles suggested some within-subject variability during the study. CONCLUSIONS: This type of biologic model can be used for the quantifiable and repeatable assessment of different needle tip designs. Needles with moderate resistance and high feel were preferred.

Lower lobe collapse during continuous interscalene brachial plexus local anesthesia at home.

OBJECTIVES: We report a case of pulmonary left lower lobe collapse following an interscalene local anesthetic infusion administered at home. This case highlights the need for patient education and postoperative communication. CASE REPORT: report A 52-year-old male patient presented for a rotator cuff repair. He was a chronic tobacco abuser with a history of occasional chest pain of unexplained cause. An interscalene catheter was placed preoperatively and surgery was performed under a combination of an interscalene block and a general anesthetic. An infusion of 0.2% ropivacaine was started via the interscalene catheter postoperatively and continued at home following his discharge from the hospital on the third postoperative day. Within 24 hours of discharge, he was readmitted to the hospital after complaining of chest pain and dyspnea. The patient was seen in the emergency department by nonanesthesiologists who were not familiar with the potential for interscalene blocks to cause diaphragmatic paresis. CONCLUSIONS: Good communication must be maintained with the patient at all times. Doctors from other specialties may be unaware of the potential complications of an interscalene block.