Ferroptosis in Haematological Malignancies and Associated Therapeutic Nanotechnologies.

Haematological malignancies are heterogeneous groups of cancers of the bone marrow, blood or lymph nodes, and while therapeutic advances have greatly improved the lifespan and quality of life of those afflicted, many of these cancers remain incurable. The iron-dependent, lipid oxidation-mediated form of cell death, ferroptosis, has emerged as a promising pathway to induce cancer cell death, particularly in those malignancies that are resistant to traditional apoptosis-inducing therapies. Although promising findings have been published in several solid and haematological malignancies, the major drawbacks of ferroptosis-inducing therapies are efficient drug delivery and toxicities to healthy tissue. The development of tumour-targeting and precision medicines, particularly when combined with nanotechnologies, holds potential as a way in which to overcome these obstacles and progress ferroptosis-inducing therapies into the clinic. Here, we review the current state-of-play of ferroptosis in haematological malignancies as well as encouraging discoveries in the field of ferroptosis nanotechnologies. While the research into ferroptosis nanotechnologies in haematological malignancies is limited, its pre-clinical success in solid tumours suggests this is a very feasible therapeutic approach to treat blood cancers such as multiple myeloma, lymphoma and leukaemia.

Integrating basophil activation tests into evaluation of perioperative anaphylaxis to neuromuscular blocking agents.

BACKGROUND: Neuromuscular blocking agents (NMBAs) remain the leading cause of perioperative anaphylaxis in Australia. Standard evaluation comprises history, skin tests, and in vitro specific immunoglobulin E tests. Drug provocation tests to suspected NMBA culprits are associated with a significant risk. Basophil activation testing (BAT) is a potentially useful in vitro test that is not commercially available in Australia or as part of standard evaluation. METHODS: All patients attending the Anaesthetic Allergy Clinic in Sydney, Australia between May 2017 and July 2018 exposed to an NMBA before the onset of anaphylaxis during their anaesthetic qualified for the study. We recruited 120 patients sequentially who received standard evaluation plus BAT using CD63, CD203c, and CD300a as surface activation markers. RESULTS: BAT results were expressed as % upregulation above the negative control and stimulation index (mean fluorescence index of stimulated sample divided by the negative control). We calculated cut-offs of 4.45% and 1.44 for CD63, and 8.80% and 1.49 for CD203c, respectively. Sensitivity was 77% with specificity of 76%. A subgroup of 10 patients with NMBA anaphylaxis had no sensitisation on skin tests. BAT using CD63 and CD203c showed sensitisation in six of these 10, and adding CD300a identified sensitisation in nine patients. BAT was positive in seven of nine patients with anaphylaxis of unknown aetiology. CONCLUSIONS: BAT may be a useful supplement to the standard evaluation in diagnosing NMBA anaphylaxis in patients with suggestive histories, but no sensitisation on skin tests. Ongoing study of this specific group of patients is required to clarify its utility in clinical practice.

Assessing cross-reactivity to neuromuscular blocking agents by skin and basophil activation tests in patients with neuromuscular blocking agent anaphylaxis.

BACKGROUND: Following diagnosis of neuromuscular blocking agent (NMBA) anaphylaxis, identifying safe alternatives for subsequent anaesthesia is critical. A patient with anaphylaxis to one NMBA can also have an allergic reaction to other NMBAs (cross-reactivity). Whilst drug provocation testing is standard for identifying or excluding allergy, there is significant risk. In vitro, after an allergen activates basophils, basophils express surface activation markers that can be measured by basophil activation testing (BAT). We compared cross-reactivity between NMBAs assessed by BAT against that by skin testing. METHODS: All patients attending an anaesthetic allergy clinic in Sydney, Australia between May 2017 and July 2018 diagnosed with NMBA anaphylaxis qualified for this study comparing intradermal skin tests and BAT with a panel of NMBAs (rocuronium, vecuronium, pancuronium, suxamethonium, cisatracurium). RESULTS: Of the 61 patients participating, sensitisation on skin testing and on BAT completely matched in only nine patients (15%). Sensitisation was not in agreement for pancuronium, cisatracurium and rocuronium, but was in agreement for vecuronium and suxamethonium. Nine patients with negative skin tests subsequently tolerated cisatracurium, and one false positive on BAT to cisatracurium was detected. CONCLUSIONS: The utility of BAT in identifying safe NMBAs for subsequent anaesthesia needs further evaluation. BAT detects a different cross-reactivity profile to skin tests. Negative skin testing and BAT might increase confidence in performing drug provocation testing, but this and follow-up of subsequent anaesthesia in our cohort is necessary to determine the clinical significance of BAT sensitisation.

Effect of extended hours dialysis on sleep quality in a randomized trial.

AIM: Poor sleep quality is common in haemodialysis patients and associated with worse outcomes. In this pre-specified analysis, we examined the impact of extended hours haemodialysis on sleep quality. METHODS: The ACTIVE Dialysis trial randomized 200 participants to extended (≥24 h/week) or standard (target 12-15 h) hours haemodialysis over 12 months. Sleep quality was measured in the Kidney Disease Quality of Life Short Form 1.3 (KDQOL-SF) by overall sleep quality score (0-10, 10 = 'very good') and the sleep subscale (0-100, 100 = 'best possible sleep') every 3 months via blinded telephone interview. The average intervention effect was calculated by mixed linear regression adjusted by time point and baseline score. Factors predicting sleep quality were assessed by multivariate regression analysis. RESULTS: Overall sleep quality score and sleep subscale at baseline were similar in both groups (5.9 [95%CI 5.4-6.4] vs. 6.3 [5.9-6.8]; 65.0 [60.9-69.1] vs. 63.2 [59.1-67.3]; extended and standard hours, respectively). Extended hours haemodialysis led to a non-significant improvement in overall sleep quality score (average intervention effect 0.44 (-0.01 to 0.89), P = 0.053) and sleep subscale (average intervention effect 3.58 (-0.02 to 7.18), P = 0.051). Poor sleep quality was associated with being female and with current smoking. Sleep quality was positively associated with EuroQol-5D (EQ5D) and the SF-36 Physical Component and Mental Component Summary Scores but not with hospitalizations. CONCLUSION: Sleep quality was not significantly improved by extended hours dialysis in this study. Sleep quality is positively correlated with quality of life in haemodialysis patients and is poorer in women and current smokers.

The Role of Cholesterol in Chronic Lymphocytic Leukemia Development and Pathogenesis.

Cholesterol has many critical functions in cells. It is a key component of membranes and cell-signalling processes, and it functions as a chemical precursor in several biochemical pathways, such as Vitamin D and steroid synthesis. Cholesterol has also been implicated in the development and progression of various cancers, in which it is thought to promote cell proliferation, migration, and invasion. Chronic lymphocytic leukemia (CLL) is an example of a lipid-avid cancer that relies on lipid metabolism, rather than glycolysis, to fuel cell proliferation. However, data regarding the role of cholesterol in CLL are conflicting. Studies have shown that dyslipidaemia is more common among CLL patients than age-matched healthy controls, and that CLL patients who take cholesterol-lowering drugs, such as statins, appear to have improved survival rates. Therefore, defining the roles of cholesterol in CLL may highlight the importance of monitoring and managing hyperlipidaemia as part of the routine management of patients with CLL. In this review, we discuss the roles of cholesterol in the context of CLL by examining the literature concerning the trafficking, uptake, endogenous synthesis, and intracellular handling of this lipid. Data from clinical trials investigating various classes of cholesterol and lipid-lowering drugs in CLL are also discussed.

Index grade group is superior to composite grade group for prediction of biochemical recurrence following radical prostatectomy.

The pathological grade of prostate cancer is the strongest predictor of recurrence. It is unclear whether the better predictor is the composite of all carcinomas within the prostate, or the highest grade lesion (index). The purpose of this study was to determine whether composite or index grade group better predicts biochemical recurrence (BCR). We undertook a retrospective analysis from a prospective institutional cohort study of men who underwent radical prostatectomy for localised prostate cancer between 2009 and 2020, in which an index and composite grade group was reported. The index grade in this study was defined as the highest grade of any tumour, usually with the highest stage, regardless of volume. Multivariate analysis and Kaplan-Meier plots were utilised. A total of 2024 men underwent radical prostatectomy during the study period; we analysed 1605 with composite grade group 2 or 3 prostate cancer. Median preoperative prostate specific antigen (PSA) was 5.9 ng/L, mean follow up was 56.8 months, 54% were pT2, 76% had multifocal disease and 16% had discordant index and composite grades. Patients with discordant index grade group had a higher risk of BCR [hazard ratio (HR) 2.22, p<0.0001]. The prevalence of BCR in the discordant group was higher at 1, 3, 5 and 7 years (4.7% vs 8.9%, 8.3% vs 18.1%, 14.5% vs 28.8% and 22.5% vs 49.5%, respectively). In cases of discordance, a higher index grade group is associated with increased rates of BCR after radical prostatectomy. Index rather than composite grade group should be used to counsel men post-operatively regarding prognosis and follow-up.

Lipid uptake in chronic lymphocytic leukemia.

Many cancers rely on glucose as an energy source, but it is becoming increasingly apparent that some cancers use alternate substrates to fuel their proliferation. Chronic lymphocytic leukaemia (CLL) is one such cancer. Through the use of flow cytometry and confocal microscopy, low levels of glucose uptake were observed in the OSU-CLL and HG3 CLL cell lines relative to highly glucose-avid Raji cells (Burkitt's lymphoma). Glucose uptake in CLL cells correlated with low expression of the GLUT1 and GLUT3 receptors. In contrast, both CLL cell lines and primary CLL cells, but not healthy B cells, were found to rapidly internalise medium- and long-chain, but not short-chain, fatty acids (FAs). Differential FA uptake was also observed in primary cells taken from patients with unmutated immunoglobulin heavy variable chain usage (IGHV) compared with patients with mutated IGHV. Delipidation of serum in the culture medium slowed the proliferation and significantly reduced the viability of OSU-CLL and HG3 cells, effects that were partially reversed by supplementation with a chemically defined lipid concentrate. These observations highlight the potential importance of FAs in the pathogenesis of CLL and raise the possibility that targeting FA utilisation may represent a novel therapeutic and prognostic approach in this disease.

Second primary malignancies in chronic lymphocytic leukaemia: Skin, solid organ, haematological and Richter's syndrome.

Chronic lymphocytic leukaemia (CLL) is invariably accompanied by some degree of immune failure, and CLL patients have a high rate of second primary malignancy (SPM) compared to the general population. We comprehensively documented the incidence of all forms of SPM including skin cancer (SC), solid organ malignancy (SOM), second haematological malignancy (SHM) and separately Richter's syndrome (RS) across all therapy eras. Among the 517 CLL/small lymphocytic lymphoma (SLL) patients, the overall incidence of SPMs with competing risks was SC 31.07%, SOM 25.99%, SHM 5.19% and RS 7.55%. Of the 216 treated patients, 106 (49.1%) had at least one form of SPM, and 63 of 106 (29.2% of treated patients) developed an SPM 1.5 years (median) after treatment for their CLL. Melanoma accounted for 30.3% of SC. Squamous cell carcinoma (SCC), including eight metastatic SCCs, was 1.8 times more than basal cell carcinoma (BCC), a reversal of the typical BCC:SCC ratio. The most common SOMs were prostate (6.4%) and breast (4.5%). SHM included seven acute myeloid leukaemia (AML) and five myelodysplasia (MDS) of which eight (four AML, four MDS) were therapy-related. Any SPM occurred in 32.1% of 53 Monoclonal B-lymphocytosis (MBL) patients. Age-adjusted standardised rates of SPM (per 100,000) for CLL, MBL and the general Australian population were 2648, 1855 and 486.9, respectively. SPMs are a major health burden with 44.9% of CLL patients with having at least one SPM, and apart from SC, associated with significantly reduced overall survival. Dramatic improvements in CLL treatment and survival have occurred with immunochemotherapy and targeted therapies, but mitigating SPM burden will be important to sustain further progress.

The future of laboratory testing in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a malignant lymphoproliferative disorder characterised by the accumulation of dysfunctional B-lymphocytes in the blood and lymphoid tissues. It is a clonally complex disease with a high degree of both intra-tumoural and inter-patient heterogeneity. This variability leads to a wide range of clinical outcomes and highlights the critical need for accurate prognostic tests in CLL. With the advent of a range of new targeted agents for CLL in recent years, there is also a clinical need for improved predictive tests to therapy. This review of laboratory testing in CLL focuses on emerging technologies for prognostication including single nucleotide polymorphism microarray for karyotypic analysis, targeted next generation sequencing analysis of the immunoglobulin heavy chain variable region gene as well as genes recurrently mutated in the disease such as TP53, and detection of minimal residual disease.

COVID-19 had no impact on emergency urological admissions at an Australian tertiary hospital.

BACKGROUNDS: The COVID-19 pandemic is an unprecedented threat to health and healthcare systems. There is no published data on the impact on urological presentations in Australia. METHODS: A retrospective analysis of all admissions under the urology service at Liverpool Hospital, Australia from February 1st to April 30th for 2020 and the previous 5 years. RESULTS: There was a total of 397 admissions in 2020 and 438 in 2019. The mean age, proportion of male, and mean length of stay were similar. In 2020, there were 229 emergency admissions. Over the same period during the previous 5 years, there were between 195 and 218 emergency admissions. In 2019, there were 220 planned admissions and 168 in 2020. Between 2019 and 2020, there was no significant difference in the proportion of patients with admission longer than 10 days (P = 0.602), requiring intensive care unit admission (P = 0.708) or inpatient operative management (P = 0.171). Among the emergency admissions, the mean Charlson Comorbidity Index was significantly lower in 2020 compared to 2019 (P = 0.009). CONCLUSIONS: Despite the pervasive fear of the COVID-19 pandemic and multiple, substantial alterations to hospital systems, structures and elective operating restrictions, no significant difference in numbers or acuity of emergency admissions were observed. Due to limitations in elective operating, there was an expected reduction in planned admissions. Our findings are in contrast to multiple recent studies and may be the result of our patient demographic where health-seeking behaviours appear to have not been significantly influenced by the pandemic.

Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses.

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a 'liquid biopsy' to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.

The ClpP activator ONC-212 (TR-31) inhibits BCL2 and B-cell receptor signaling in CLL.

Despite advances in therapy, a significant proportion of patients with chronic lymphocytic leukemia (CLL) relapse with drug resistant disease. Novel treatment approaches are required, particularly for high risk disease. The imipridones represent a new class of cancer therapy that has been investigated in pre-clinical and clinical trials against a range of different cancers. We investigated the effects of the imipridone, ONC-212, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment and a TP53ko CLL cell line (OSU-CLL-TP53ko). ONC-212 induced dose-dependent apoptosis, cell cycle arrest and reduced the migration of CLL cells in vitro, including cells from patients with TP53 lesions and OSU-CLL-TP53ko cells. The effects of ONC-212 were associated with protein changes consistent with activation of the mitochondrial protease, CIpP, and the integrated stress response. We also observed inhibition of pathways downstream of the B-cell receptor (BCR) (AKT and MAPK-ERK1/2) and a pro-apoptotic shift in the balance of proteins of the BCL2 family of proteins (BCL2, MCL1, BCLxL, BAX and NOXA). In conclusion, the study suggests ONC-212 may represent an effective treatment for high risk CLL disease by inhibiting multiple facets of the BCR signaling pathway and the pro-survival effects of the BCL2-family proteins.

Therapeutic approaches and drug-resistance in chronic lymphocytic leukaemia.

The treatment of chronic lymphocytic leukaemia has been revolutionised in recent years, first by the introduction of chemoimmunotherapy regimens and subsequently by the development of drugs, including ibrutinib, idelalisib and venetoclax, that target components of the B-cell receptor signalling pathway or B-cell lymphoma 2 family of proteins. Despite high initial response rates in patients treated with chemoimmunotherapy or targeted agents, a significant proportion of patients relapse with progressive and refractory disease. In a subset of these patients, drug resistance has been associated with specific genetic lesions or activation of alternate pro-survival pathways. However, the mechanisms that confer drug resistance in the remainder of the patients with refractory disease have yet to be fully elucidated. In this review, we discuss our current understanding of the mechanics of drug resistance in chronic lymphocytic leukaemia and describe how this knowledge may aid in rationalising future treatment strategies to prevent the development of refractory or aggressive transformation of the disease.

Fatty acid synthase and adenosine monophosphate-activated protein kinase regulate cell survival and drug sensitivity in diffuse large B-cell lymphoma.

Fatty acid synthesis is crucial in supporting the survival and proliferation of multiple forms of cancer. The high metabolic demands of fatty acid synthesis are regulated by the AMP-activated kinase and activity of the fatty acid synthase enzyme. In this study, the roles of these enzymes in diffuse large B-cell lymphoma (DLBCL) were investigated by genetic knock-down and pharmacological activation of AMP-activated kinase by metformin, and selective inhibition of fatty acid synthase using the novel drug Fasnall. We observed distinct heterogeneity and adaptive plasticity of lipid metabolism in a panel of DLBCL cell lines and demonstrate the therapeutic potential of inhibiting fatty acid synthesis in a subset of DLBCL cells. The translational relevance of these in vitro data is supported by the strong correlation between AMP-activated protein kinase expression in primary DLBCL samples and disease relapse. Inhibition of fatty acid synthase with Fasnall may represent a therapeutic option for DLBCL that preferentially subverts to de novo fatty acid synthesis.

The phosphorylation status of membrane-bound nucleoside diphosphate kinase in epithelia and the role of AMP.

Nucleoside diphosphate kinase (NDPK) has many roles and is present in different locations in the cell. Membrane-bound NDPK is present in epithelial fractions enriched for the apical membrane. Here, we show in human, mouse and sheep airway membranes, that the phosphorylation state of membrane-bound NDPK on histidine and serine residues differs dependent on many regulatory factors. GTP (but not ATP) promotes serine phosphorylation (pSer) of NDPK. Further we find that rising [AMP] promotes pSer (only with GTP) but inhibits histidine phosphorylation (pHis) of NDPK from both donors. We find that NDPK co-immunoprecipitates reciprocally with AMP-activated kinase and that these two proteins can co-localise in human airways. AMP concentrations rise rapidly when ATP is depleted or during hypoxia. We find that, in human airway cells exposed to hypoxia (3% oxygen), membrane-bound NDPK is inhibited. Although histidine phosphorylation should in principle be independent of the nucleotide triphosphates used, we speculate that this membrane pool of NDPK may be able to switch function dependent on nucleotide species.

National trends in urinary diversion over the past 20 years: an Australian study.

BACKGROUND: To investigate the trends in urinary diversion (UD) in Australia over the past 20 years, to correlate with patient demographics and to compare with international data. METHODS: A retrospective analysis of Medicare Australia data was performed using the relevant Medicare Benefit Schedule procedure codes over the past 20 years. Included diversion procedures were ureterocutaneous, ureterocolonic, intestinal conduit and continent reservoir. All patients aged older than 15 years were included in the analyses. RESULTS: Over the past two decades, 6124 cystectomies and 7166 UDs were subsidized by Medicare Australia. The median age group for UD was 65-74 years old and 71.8% were male. Intestinal conduit accounted for the majority of UDs (84.9%), followed by continent reservoirs (11.8%). Ureterocolonic and ureterocutaneous accounted for small proportions (2.9% and 0.4%, respectively). The absolute numbers of UD procedures increased over the past 20 years but the proportion of different methods remained constant. The rates of continent reservoir UD were significantly higher in men and people aged less than 55 years old (P < 0.001 for both). Over the course of the study, the proportion of people aged greater than 75 years undergoing UD increased significantly (P < 0.001). CONCLUSION: In contrast to major international academic institutions, the proportion of continent reservoir UDs performed in Australia has not changed over the past two decades. Intestinal conduit remains the most common UD procedure.

Associations of Impaired Renal Function With Declines in Muscle Strength and Muscle Function in Older Men: Findings From the CHAMP Study.

BACKGROUND: Advanced kidney disease is associated with reduced muscle strength and physical performance. However, associations between early stages of renal impairment and physical outcomes are unclear. METHODS: The Concord Health and Ageing in Men Project is a prospective study of 1,705 community-dwelling men aged 70 years and older. Participants with estimated glomerular filtration rate (eGFR) more than 30 mL/min/1.73 m2 were included and further divided into four eGFR categories. Physical parameters including grip strength, gait speed, appendicular lean mass (ALM, a sum of skeletal mass of arms and legs), ALM adjusted for body mass index (ALMBMI), and muscle function (measured using grip strength divided by arm lean mass) were assessed at both baseline and 5-year follow-up. Associations between kidney function and changes in physical parameters were analyzed using linear and logistic regression models. RESULTS: Our study included 789 men with a median age of 75 years and median eGFR of 72 mL/min/1.73 m2 at baseline. Over 5 years, grip strength, gait speed, ALMBMI, and muscle function all declined in the whole cohort, compared with baseline. The multivariable analyses showed that poorer renal function was associated with more rapid declines in grip strength, gait speed, and muscle function in participants with mild-to-moderate renal impairment (GFR category stage G3, eGFR < 60 mL/min/1.73 m2) (p = .01, p < .01, p = .02, respectively) but less so in those with eGFR more than 60 mL/min/1.73 m2, whereas eGFR category did not have a significant impact on declines in ALMBMI. These results remained unchanged with or without adjustment for age. CONCLUSIONS: In community-dwelling older men, mild-to-moderate renal impairment at baseline was associated with declines in grip strength, gait speed, and muscle function over time despite preservation of muscle mass.