### Recent Submissions

• #### Laboratory methods for monitoring argatroban in heparin-induced thrombocytopenia

Introduction: The Summary of Product Characteristics for the direct thrombin inhibitor argatroban states monitoring should be by activated partial thromboplastin time (APTT), with a target range of 1.5-3.0 times the patients' baseline APTT. APTT may be influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. Previous studies have shown sensitivity differences of APTT reagents to argatroban. Some recent publications have favoured the use of anti-IIa methods to determine the plasma drug concentration of argatroban. This study aims to compare the anti-IIa assays: Hemoclot thrombin inhibitor assay (HTI) and Ecarin chromogenic assay (ECA) alongside the APTT. Methods: Residual plasma of 25 samples from 8 patients (3 with COVID-19 and HIT: n = 18, 5 with HIT: n = 7) was tested at two sites: site A: Sysmex CS5100 by HTI and APTT (Actin FS and SynthASil), and also on Stago STA Compact Max: ECA and APTT (CK Prest); and site B: Stago STA R Max 2 by ECA and APTT (Cephascreen). Results: Mean APTT ratio was 1.96 (Actin FS), 1.84 (SynthASil), 1.59 (CK Prest) and 2.48 (Cephascreen). Mean argatroban concentration by HTI was 0.60 µg/mL and by ECA was 0.65 µg/mL (site A) and 0.70 µg/mL (site B). There was a poor correlation to HTI in APTT ratios (range r2 = .0235-0.4181) with stronger correlations between ECA methods to HTI (r2 = .8998 site A, r2 = .8734 site B). Conclusion: This study confirms previous publications on the unsuitability of APTT and expands this by being multisited and included APTT reagents that use mechanical clot detection. Both anti-IIa methods are more suitable.
• #### The role of 4-Dimensional flow in the assessment of bicuspid aortic valve and its valvulo-aortopathies

Bicuspid aortic valve is the most common congenital cardiac malformation and the leading cause of aortopathy and aortic stenosis in younger patients. Aortic wall remodelling secondary to altered haemodynamic flow patterns, changes in peak velocity, and wall shear stress may be implicated in the development of aortopathy in the presence of bicuspid aortic valve and dysfunction. Assessment of these parameters as potential predictors of disease severity and progression is thus desirable. The anatomic and functional information acquired from 4D flow MRI can allow simultaneous visualisation and quantification of the pathological geometric and haemodynamic changes of the aorta. We review the current clinical utility of haemodynamic quantities including velocity, wall sheer stress and energy losses, as well as visual descriptors such as vorticity and helicity, and flow direction in assessing the aortic valve and associated aortopathies.
• #### Infliximab: a single-centre, prospective, observational evaluation of TDM data in patients with IBD

Objectives: Therapeutic drug monitoring of infliximab (IFX) is important to optimise treatment of inflammatory bowel disease (IBD). A recent IBD consensus statement recommends targeting trough serum concentrations of >3 μg/mL, higher than our local recommendation of >1 μg/mL. We therefore investigated the relationship between IFX trough concentrations and C reactive protein (CRP), faecal calprotectin (FCP), clinical outcomes and anti-IFX antibody (AB) development as well as the influence of concomitant thiopurine treatment. Methods: Observational data, prospectively collected in a cohort of adult patients with IBD newly initiated on IFX at a single centre. Results: IFX concentrations >3 μg/mL were associated with a greater reduction in CRP (% change from baseline) and lower FCP; mean (SD) 47 (33.8) % vs 102.3 (136.9) % and 233.9 (505.1) μg/g vs 416.3 (613.5) μg/g, respectively. Lower IFX concentrations were observed in patients who developed AB than those who did not, mean (range) 6.2 (1.1-10) μg/mL vs 0.9 (0.4-4.9) μg/mL, respectively, and also in patients who stopped/switched therapy compared with those who continued, 2.4 (2.9) μg/mL vs 6.5 (2.8) μg/mL; p=0.0002. Patients taking a concomitant thiopurine were found to have higher IFX concentrations; mean (range) 6.4 (0.7-10) μg/mL vs 3.9 (0.4-10) μg/mL. Conclusions: IFX concentrations are correlated with biomarkers, clinical response and AB development in patients with IBD. Concomitant thiopurine therapy appears to be associated with higher IFX concentrations and reduced likelihood of AB development.
• #### Comparison of the prognostic value of microscopically measured invasive width versus macroscopic width in cutaneous melanoma shows the superiority of microscopic invasive width measurement

Background: Invasive width, the distance between the most peripheral invasive melanoma cells on the section where Breslow thickness (BT) was measured, was recently identified as a prognostic feature. It is unclear whether a routine measurement is justified, given that macroscopic width is already included in many melanoma histopathology reports and may itself be a prognostic feature. This study sought to investigate this. Methods: A retrospective cohort of 718 melanoma patients in which macroscopic width had been stated in the original histopathology report was used. Survival analysis was performed. Results: Macroscopic and invasive widths were positively correlated (p < 0.001). Invasive width was typically smaller than the paired macroscopic width (median difference 3.7 mm, p < 0.001), a difference seen across all T groups. Both macroscopic and invasive widths were significantly associated with melanoma survival in Kaplan-Meier analysis, including overall survival, but invasive width survival curves were more widely separated. Both were significantly associated with outcome after correction for BT in Cox proportional hazards regression, but the models containing invasive width had a substantially better fit. Conclusions: This study shows that both macroscopic and invasive widths have prognostic values, but confirms that the latter is superior. It supports further investigation of this feature's prognostic value.
• #### Outbreak of SARS-CoV-2 at a hospice: terminated after the implementation of enhanced aerosol infection control measures

Outbreaks of COVID-19 in hospices for palliative care patients pose a unique and difficult situation. Staff, relatives and patients may be possible sources and recipients of infection. We present an outbreak of COVID-19 in a hospice setting, during the UK's first pandemic wave. During the outbreak period, 26 patients and 30 staff tested SARS-CoV-2 positive by laboratory-based RT-PCR testing. Most infected staff exhibited some mild, non-specific symptoms so affected staff members may not have voluntarily self-isolated or had themselves tested on this basis. Similarly, for infected patients, most became symptomatic and were then isolated. Additional, enhanced aerosol infection control measures were implemented, including opening of all windows where available; universal masking for all staff, including in non-clinical areas and taking breaks separately; screening for asymptomatic infection among staff and patients, with appropriate isolation (at home for staff) if infected; performing a ventilation survey of the hospice facility. After these measures were instigated, the numbers of COVID-19 cases decreased to zero over the following three weeks. This outbreak study demonstrated that an accurate understanding of the routes of infection for a new pathogen, as well as the nature of symptomatic versus asymptomatic infection and transmission, is crucial for controlling its spread.

• #### Predictors of adverse outcome in the first and second waves of the COVID-19 pandemic: results from a UK centre

Background/aims: Data concerning differences in demographics/disease severity between the first and second waves of COVID-19 are limited. We aimed to examine prognosis in patients presenting to hospital with COVID-19 amongst different ethnic groups between the first and second waves in the UK. Methods: In this retrospective cohort study, we included 1763 patients presenting to a regional hospital centre in Leicester (UK) and compared those in the first (n = 956) and second (n = 807) waves. Admission National Early Warning Scores, mechanical ventilation and mortality rate were lower in the second wave compared with the first. Results: Thirty-day mortality risk in second wave patients was approximately half that of first wave patients [adjusted hazard ratio (aHR) 0.55, 95% confidence interval (CI) 0.40-0.75]. In the second wave, Black patients were at higher risk of 30-day mortality than White patients (4.73, 1.56-14.3). Conclusion: We found that disporportionately higher risks of death in patients from ethnic minority groups were not equivalent across consecutive waves of the pandemic. This suggests that risk factors for death in those from ethnic minority groups are malleable and potentially reversible. Our findings need urgent investigation in larger studies.
• #### Incidence of synchronous contralateral tonsillar malignancy in a known case of unilateral tonsillar carcinoma

Objective: The literature is divided with regards to contralateral tonsillectomy in a known/suspected case of ipsilateral tonsillar malignancy. In this study, we evaluate the incidence of indolent synchronous contralateral tonsillar malignancy (SCTC) in patients with known ipsilateral tonsillar malignancy. Methods: All patients diagnosed with ipsilateral tonsillar carcinoma (TC) at a tertiary teaching center between January 2016 and December 2019 were screened. None of the patients were suspected to have bilateral TC. All patients underwent appropriate imaging in the form of Magnetic resonance imaging and computed tomography of head and neck region and then underwent bilateral tonsillectomy. The prevalence of bilateral tonsillar malignancy and the factors predicting them were analyzed. Results: In all 59 patients were included in the study. The mean and median age of the patient population was 60.8 and 59 years, respectively, with a male to female ratio of 3.2:1. The incidence of bilateral tonsillar malignancy in carcinoma of unknown primary (CUP) was 3/10 (33.3%). Among the remaining 49 patients, incidence of synchronous contralateral tonsillar carcinoma (SCTC) was 2/49 (4.08%). Overall, 5/59 (8.5%) patients had synchronous bilateral tonsillar malignancy. Furthermore, dysplasia was found in the contralateral tonsil in 4/10 (40%) CUP patients. Among the remaining 49 patients, dysplasia was seen in the contralateral tonsil in 20/49 (40.8%) patients. The absence of p16 expression predicted higher probability of SCTC. Factors like gender, T stage, nodal status or smoking did not predict SCTC. Conclusion: We recommend bilateral tonsillectomy in all patients with suspected or proven TC (unilateral or bilateral) and CUP as it helps identify indolent SCTC and it does not add any significant morbidity to the patients.
• #### Increased mitochondrial proline metabolism sustains proliferation and survival of colorectal cancer cells

Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells' proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells' survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.
• #### Feasibility of arterial spin labeling in evaluating high- and low-flow peripheral vascular malformations: a case series

We present a case series highlighting a novel use of arterial spin labeling (ASL), a MRI perfusion technique, to evaluate both high- and low-flow peripheral vascular malformations (PVMs) across a range of anatomical locations. While the role of ASL in assessing intracranial vascular malformations is more established, there is limited evidence for PVMs. Our results provide preliminary evidence for the feasibility of ASL in imaging PVMs and its potential ability to distinguish between high- and low-flow PVMs. In addition, we demonstrate its ability to identify focal high blood flow, which may indicate the nidus in arteriovenous malformations. Together, these findings have important implications for patient management. We also outline the potential benefits and limitations of ASL in the imaging of PVMs, and provide justification for further validation of its diagnostic performance.
• #### Toxic epidermal necrolysis-like lupus

Toxic epidermal necrosis (TEN)-like lupus is a rare condition characterized by epidermal loss and mucosal ulceration occurring in patients with acute severe flares of systemic lupus erythematosus. The clinical picture may mimic drug-induced Stevens-Johnson syndrome/TEN; however, the absence of a suitable culprit drug, and the context of acute lupus point to the correct diagnosis. In a case series of three patients, further discriminating features included a slower onset of epidermal loss, more limited mucosal ulceration and a lack of ocular involvement when compared with drug-induced TEN. Histology may show similar features, including basal layer vacuolation, apoptosis and full-thickness epidermal necrosis. Patients with TEN-like lupus may have additional features of lupus, and a lupus band on direct immunofluorescence. It is important to identify this condition correctly, so that these patients can be appropriately managed with early input from Rheumatologists and prompt treatment with high-dose combined immunosuppressant therapy.
• #### Evaluation of an 8-Week vegan diet on plasma trimethylamine-N-oxide and postchallenge glucose in adults with dysglycemia or obesity

Background: Trimethylamine N-oxide (TMAO), a metabolite generated by the gut in response (in part) to meat consumption, is linked to poor cardiometabolic health. Objectives: We investigate the effect of an 8-week vegan diet, followed by a 4-week period of unrestricted diet, on glucose tolerance and plasma TMAO in human omnivores with obesity or dysglycemia. Methods: This interventional single-group prospective trial involved 23 regular meat eaters with dysglycemia [glycated hemoglobin ≥ 5.7% and ≤8% (39-64 mmol/mol)], or obesity (ΒΜΙ ≥ 30 kg/m2) aged 57.8 ± 10.0 years. Participants [14 men (60.9%) and 9 women (39.1%)] were supported in following a vegan diet for 8 weeks, followed by 4 weeks of unrestricted diet. The primary outcomes (plasma TMAO and glucose) were assessed at baseline, during the vegan diet (weeks 1 and 8), and after the unrestricted diet period (week 12). TMAO was assessed after fasting and glucose was measured as a time-averaged total AUC using a 180-minute oral-glucose-tolerance test. Generalized estimating equation models with an exchangeable correlation structure were used to assess changes from baseline, adjusting for age, sex, ethnicity, and weight. Results: TMAO levels (marginal mean) were reduced after weeks 1 and 8 of a vegan diet compared to baseline, from 10.7 (97.5% CI, 6.61-17.3) μmol/L to 5.66 (97.5% CI, 4.56-7.02) μmol/L and 6.38 (97.5% CI, 5.25-7.74) μmol/L, respectively; however, levels rebounded at week 12 after resumption of an unrestricted diet (17.5 μmol/L; 97.5% CI, 7.98-38.4). Postprandial glucose levels (marginal means) were reduced after weeks 1 and 8 compared to baseline, from 8.07 (97.5% CI, 7.24-8.90) mmol/L to 7.14 (97.5% CI, 6.30-7.98) mmol/L and 7.34 (97.5% CI, 6.63-8.04) mmol/L, respectively. Results for glucose and TMAO were independent of weight loss. Improvements in the lipid profile and markers of renal function were observed at week 8. Conclusions: These findings suggest that a vegan diet is an effective strategy for improving glucose tolerance and reducing plasma TMAO in individuals with dysglycemia or obesity. This study was registered at clinicaltrials.gov as NCT03315988.
• #### MNK inhibition sensitizes KRAS-mutant colorectal cancer to mTORC1 inhibition by reducing eIF4E phosphorylation and c-MYC expression

KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.
• #### Inflammation and physical dysfunction: responses to moderate intensity exercise in chronic kidney disease

Background: People with chronic kidney disease (CKD) experience skeletal muscle wasting, reduced levels of physical function and performance, and chronic systemic inflammation. While it is known that a relationship exists between inflammation and muscle wasting, the association between inflammation and physical function or performance in CKD has not been well studied. Exercise has anti-inflammatory effects, but little is known regarding the effect of moderate intensity exercise. This study aimed to (i) compare systemic and intramuscular inflammation between CKD stage G3b-5 and non-CKD controls; (ii) establish whether a relationship exists between physical performance, exercise capacity and inflammation in CKD; (iii) determine changes in systemic and intramuscular inflammation following 12 weeks of exercise; and (iv) investigate whether improving inflammatory status via training contributes to improvements in physical performance and muscle mass. Methods: This is a secondary analysis of previously collected data. CKD patients stages G3b-5 (n = 84, n = 43 males) and non-CKD controls (n = 26, n = 17 males) underwent tests of physical performance, exercise capacity, muscle strength and muscle size. In addition, a subgroup of CKD participants underwent 12 weeks of exercise training, randomized to aerobic (AE, n = 21) or combined (CE, n = 20) training. Plasma and intramuscular inflammation and myostatin were measured at rest and following exercise. Results: Tumour necrosis factor-α was negatively associated with lower $^{^{^{.}}}{\rm V}$O2Peak (P = 0.01), Rectus femoris-cross sectional area (P = 0.002) and incremental shuttle walk test performance (P < 0.001). Interleukin-6 was negatively associated with sit-to-stand 60 performances (P = 0.006) and hand grip strength (P = 0.001). Unaccustomed exercise created an intramuscular inflammatory response that was attenuated following 12 weeks of training. Exercise training did not reduce systemic inflammation, but AE training did significantly reduce mature myostatin levels (P = 0.02). Changes in inflammation were not associated with changes in physical performance. Conclusions: Systemic inflammation may contribute to reduced physical function in CKD. Twelve weeks of exercise training was unable to reduce the level of chronic systemic inflammation in these patients, but did reduce plasma myostatin concentrations. Further research is required to further investigate this.
• #### Accidental neutron exposure in a medical setting: a case study

In May 2016, a new linear accelerator (Linac) was installed at a hospital oncology department. A team of individuals supervised the installation, including a Radiation Oncologist who acted as an independent observer to the installation, calibration, beam data collection and shielding measurements. In order to ensure the shielding was correct, a licensed representative of the Turkish Atomic Energy Authority carried out formal measurements of the gamma and neutron dose rates at a variety of locations in and around the Linac facility. At 18 MV, the maximum neutron dose rate was 172μSv h-1and the maximum gamma dose rate was approximately 2μSv h-1(ambient dose equivalent in both cases), significantly higher than the expected and local background doses. As the neutron dose rates in particular were so high, it was concluded that the shielding was not sufficient, potentially due to an inadequate design. In order to rule out overexposure during the installation, biological dosimetry was carried out for a number of the individuals involved. The estimated doses were closely aligned with the doses measured using commercially available neutron dosemeters and were also within the tolerance dose ranges estimated using Monte Carlo simulations, which also supported the investigation. The results underline the need for careful planning before and after installation of new radiation exposure facilities, especially high MV Linac operation for which photo-neutrons might need to be mitigated. The results clearly indicate the importance of such checks, in addition to demonstrating the relevance of biological dosimetry supported by modelling strategies complex or unclear exposure scenarios.
• #### NUT carcinoma arising from the parotid gland: a case report and review of the literature

NUT carcinoma is an aggressive carcinoma with an overall poor survival outcome. The mediastinum and head and neck area, especially the sinonasal region, are among the common sites of disease. Histopathological diagnosis of NUT carcinoma is often very challenging due to its overlapping features with other poorly differentiated carcinomas. We report a case of NUT carcinoma arising from the parotid gland of a young female patient. Primary NUT carcinoma of salivary gland is very rare, with only 15 such cases reported in the literature to date. Our case highlights the diagnostic challenges associated with such lesions.
• #### Nonadherence in hypertension: how to develop and implement chemical adherence testing

Nonadherence to antihypertensive medication is common, especially in those with apparent treatment-resistant hypertension (true treatment-resistant hypertension requires exclusion of nonadherence), and its routine detection is supported by clinical guidelines. Chemical adherence testing is a reliable and valid method to detect adherence, yet methods are unstandardized and are not ubiquitous. This article describes the principles of chemical adherence testing for hypertensive patients and provides a set of recommendations for centers wishing to develop the test. We recommend testing should be done in either of two instances: (1) in those who have resistant hypertension or (2) in those on 2 antihypertensives who have a less than 10 mm Hg drop in systolic blood pressure on addition of the second antihypertensive medication. Furthermore, we recommend that verbal consent is secured before undertaking the test, and the results should be discussed with the patient. Based on medications prescribed in United Kingdom, European Union, and United States, we list top 20 to 24 drugs that cover >95% of hypertension prescriptions which may be included in the testing panel. Information required to identify these medications on mass spectrometry platforms is likewise provided. We discuss issues related to ethics, sample collection, transport, stability, urine versus blood samples, qualitative versus quantitative testing, pharmacokinetics, instrumentation, validation, quality assurance, and gaps in knowledge. We consider how to best present, interpret, and discuss chemical adherence test results with the patient. In summary, this guidance should help clinicians and their laboratories in the development of chemical adherence testing of prescribed antihypertensive drugs.
• #### Phosphaturic mesenchymal tumors: radiological aspects and suggested imaging pathway

Phosphaturic mesenchymal tumors (PMTs) are rare mesenchymal neoplasms of soft tissue or bone origin that can give rise to a challenge in diagnostic imaging. These tumors are frequently associated with tumor-induced osteomalacia, also called oncogenic osteomalacia, which is a rare paraneoplastic syndrome characterized by ectopic secretion of fibroblast growth factor 23, a hormone that regulates serum phosphate level. PMTs show polymorphic features on both radiological findings and histological examination, causing problems in diagnosis owing to their similarity with other mesenchymal tumors. Thus, this paper aims to describe radiological aspects of PMTs and suggest an imaging pathway for accurate diagnosis throughout the evidence from the literature review.
• #### Severe symptomatic hypercalcemia in a patient with familial hypocalciuric hypercalcemia

One of the less common causes of hypercalcemia is familial hypocalciuric hypercalcemia (FHH). It is an autosomal-dominant genetic condition, which presents asymptomatically in most patients while some may have mild symptoms. The serum calcium levels are mildly elevated with mild elevation in parathyroid hormone, which rarely requires management with pharmacologic agents. We present an unusual case report of a 76-year-old woman, confirmed to have FHH type 1 mutation, presented with symptomatic hypercalcemia probably set off by metabolic stresses of her age and needing intensive treatment with intravenous bisphosphonates, calcitonin and cinacalcet.
• #### Ultrasound shear wave elastography imaging of common carotid arteries in patients with Spontaneous Coronary Artery Dissection (SCAD)

Background: Shear wave elastography (SWE) is emerging as a valuable clinical tool for a variety of conditions. The aim of this pilot study was to assess the potential of SWE imaging of the common carotid arteries (CCA) in patients with spontaneous coronary artery dissection (SCAD), a rare but potentially life-threatening condition, hypothesized to be linked to changes in vessel wall elasticity. Methods: Ultrasound shear wave elastography (SWE) estimates of artery wall elasticity were obtained from the left and right CCAs of 89 confirmed SCAD patients and 38 non-dissection controls. SWE images obtained over multiple cardiac cycles were analysed by a blinded observer to estimate elasticity in the form of a Young's Modulus (YM) value, across regions of interest (ROI) located within the anterior and posterior CCA walls. Results: YM estimates ranged from 17 to 133 kPa in SCAD patients compared to 34 to 87 kPa in non-dissection controls. The mean YM of 55 [standard deviation (SD): 21] kPa in SCAD patients was not significantly different to the mean of 57 [SD: 12] kPa in controls, p = 0.32. The difference between groups was 2 kPa [95% Confidence Interval - 11, 4]. Conclusions: SWE imaging of CCAs in SCAD patients is feasible although the clinical benefit is limited by relatively high variability of YM values which may have contributed to our finding of no significant difference between SCAD patients and non-dissection controls.