Identification, validation and biological characterisation of novel glioblastoma tumour microenvironment subtypes: implications for precision immunotherapy.

BACKGROUND: New precision medicine therapies are urgently required for glioblastoma (GBM). However, to date, efforts to subtype patients based on molecular profiles have failed to direct treatment strategies. We hypothesised that interrogation of the GBM tumour microenvironment (TME) and identification of novel TME-specific subtypes could inform new precision immunotherapy treatment strategies. MATERIALS AND METHODS: A refined and validated microenvironment cell population (MCP) counter method was applied to >800 GBM patient tumours (GBM-MCP-counter). Specifically, partition around medoids (PAM) clustering of GBM-MCP-counter scores in the GLIOTRAIN discovery cohort identified three novel patient clusters, uniquely characterised by TME composition, functional orientation markers and immune checkpoint proteins. Validation was carried out in three independent GBM-RNA-seq datasets. Neoantigen, mutational and gene ontology analysis identified mutations and uniquely altered pathways across subtypes. The longitudinal Glioma Longitudinal AnalySiS (GLASS) cohort and three immunotherapy clinical trial cohorts [treatment with neoadjuvant/adjuvant anti-programmed cell death protein 1 (PD-1) or PSVRIPO] were further interrogated to assess subtype alterations between primary and recurrent tumours and to assess the utility of TME classifiers as immunotherapy biomarkers. RESULTS: TMEHigh tumours (30%) displayed elevated lymphocyte, myeloid cell immune checkpoint, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 transcripts. TMEHigh/mesenchymal+ patients featured tertiary lymphoid structures. TMEMed (46%) tumours were enriched for endothelial cell gene expression profiles and displayed heterogeneous immune populations. TMELow (24%) tumours were manifest as an 'immune-desert' group. TME subtype transitions upon recurrence were identified in the longitudinal GLASS cohort. Assessment of GBM immunotherapy trial datasets revealed that TMEHigh patients receiving neoadjuvant anti-PD-1 had significantly increased overall survival (P = 0.04). Moreover, TMEHigh patients treated with adjuvant anti-PD-1 or oncolytic virus (PVSRIPO) showed a trend towards improved survival. CONCLUSIONS: We have established a novel TME-based classification system for application in intracranial malignancies. TME subtypes represent canonical 'termini a quo' (starting points) to support an improved precision immunotherapy treatment approach.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

A randomized trial regarding antimicrobial prophylaxis (AMP) in transurethral resection of bladder tumor (TURB).

PURPOSE: To determine whether omitting antimicrobial prophylaxis (AMP) in TURB is safe in patients undergoing TURB without an indwelling pre-operative catheter/nephrostomy/DJ and a negative pre-operative urinary culture. MATERIALS AND METHODS: A multi-centered randomized controlled trial (RCT) from 17-09-2017 to 31-12-2019 in 5 hospitals. Patients with a pre-operative indwelling catheter/DJ-stent or nephrostomy and a positive pre-operative urinary culture (> 104 uropathogens/mL) were excluded. Post-operative fever was defined as body temperature ≥ 38.3 °C. A non-inferiority design with a 6% noninferiority margin and null hypothesis (H0) that the infection risk is at least 6% higher in the experimental (E) than in the control (C) group; H0: C (AMP-group) - E (no AMP-group) ≥ Δ (6% noninferiority margin). A multivariable, logistic regression was performed for AMP and post-TURB fever with covariates: tumor size and (clot-) retention. The R Project® for statistical computing was used for statistical analysis and a p value of 0.05 was considered as statistically significant. RESULTS: 459 Patients were included and 202/459 (44.1%) received AMP vs 257/459 (55.9%) without AMP. Fever occurred in 6/202 [2.9%; 95% CI (1.2-6.6%)] patients with AMP vs 8/257 [3.1%; 95% CI (1.5%-6.1%)] without AMP (p = 0.44). Multivariable, logistic regression showed no significant harm in omitting AMP when controlled for (clot-)retention and tumor size (p = 0.85) and an adjusted risk difference in developing post-TURB fever of 0.0016; 95% CI [- 0.029; 0.032]. CONCLUSION: Our data suggest the safety of omitting AMP in patients undergoing TURB without an indwelling, pre-operative catheter/nephrostomy/DJ and a negative pre-operative urinary culture.

Antimicrobial Prophylaxis in Transurethral Resection of the Prostate: Results of a Randomized Trial.

PURPOSE: We sought to determine whether omitting antimicrobial prophylaxis is safe in patients undergoing transurethral resection of the prostate without preoperative pyuria and a preoperative catheter. MATERIALS AND METHODS: We conducted a multicenter randomized controlled trial from September 17, 2017 until December 31, 2019 in 5 hospitals. Patients with pyuria (>100 white blood cells/ml) and a preoperative indwelling catheter were excluded. Postoperative fever was defined as a body temperature ≥38.3C. A noninferiority design was used with a 6% noninferiority margin and null hypothesis (H0) that the infection risk is at least 6% higher in the experimental (E) than in the control (C) group; H0: C (antimicrobial prophylaxis group) - E (no antimicrobial prophylaxis group) ≥ Δ (6% noninferiority margin). A multivariable, logistic regression was performed regarding posttransurethral resection of the prostate fever and antimicrobial prophylaxis with co-variates: (clot-)retention and operating time. The R Project® for statistical computing was used and a p value of 0.05 was considered as statistically significant. RESULTS: Of the patients 474 were included for multivariable analysis and 211/474 (44.5%) received antimicrobial prophylaxis vs 263/474 (55.5%) patients without antimicrobial prophylaxis. Antibiotics were fluoroquinolones in 140/211 (66.4%), cephazolin in 58/211 (27.5%) and amikacin in 13/211 (6.2%) patients. Fever occurred in 9/211 (4.4%) patients with antimicrobial prophylaxis vs 13/263 (4.9%) without antimicrobial prophylaxis (p=0.8, risk difference 0.006 [95% CI -0.003-0.06, relative risk 1.16]). We were able to exclude a meaningful increase in harm associated with omitting antimicrobial prophylaxis (p=0.4; adjusted risk difference 0.016 [95% CI -0.02-0.05]). CONCLUSIONS: Our data demonstrate the safety of omitting antimicrobial prophylaxis in patients undergoing transurethral resection of the prostate without preoperative pyuria and a preoperative indwelling catheter.

Antibiotic prophylaxis in TURP: a prospective analysis concerning antibiotic stewardship and a potential reduction of antibiotic use in TURP.

PURPOSE: Antibiotic prophylaxis is standard procedure in transurethral resection of the prostate (TURP). We evaluated the necessity of antibiotic (AB) prophylaxis in TURP due to increasing microbial antibiotic resistance. METHODS: This is a prospective cohort study of 506 patients. Only patients with a pre-operative catheter/pyuria received AB-prophylaxis. Urine analysis (pre-operative, at discharge, and 3 week post-operative) was performed next to an analysis of the blood culture/irrigation fluid and of the resected prostatic tissue. Statistical analysis was performed using Fisher's exact test. RESULTS: 67/506 (13.2%) patients received prophylactic antibiotics. 56/67 (83.5%) patients had a pre-operative catheter and 11/67 (16.4%) had pre-operative pyuria in which a fluoroquinolone-resistance (FQ-R) rate of 69.2% in Escherichia coli (EC) was observed. Clinical infectious symptoms were present in 13/439 (2.9%) patients without antibiotic prophylaxis; 12/439 (2.7%) patients had uncomplicated fever (<38.5°) during or after hospitalization and only 1/439 patient (0.2%) was high degree fever (> 38.5°) observed. Uncomplicated fever developed in 7/67 (10.4%) patients who did receive AB-prophylaxis. FQ-R was observed in 60% of the positive urine cultures at discharge and in 53.8% 3 week post-operatively. CONCLUSIONS: Our data show a low infectious complication rate (2.9%) in patients without a pre-operative catheter or pyuria,undergoing TURP without AB-prophylaxis. These findings might question the current use of AB prophylaxis in TURP in patients without a pre-operative catheter or pyuria, in times of antibiotic stewardship due to the high rate of microbial-resistance in our population.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis.

BACKGROUND AND AIMS: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor alpha (anti-TNFalpha) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. METHODS: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. RESULTS: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. CONCLUSION: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Long-term outcome of treatment with infliximab in 614 patients with Crohn's disease: results from a single-centre cohort.

BACKGROUND AND AIMS: This observational study assessed the long-term clinical benefit of infliximab (IFX) in 614 consecutive patients with Crohn's disease (CD) from a single centre during a median follow-up of 55 months (interquartile range (IQR) 27-83). METHODS: The primary analysis looked at the proportion of patients with initial response to IFX who had sustained clinical benefit at the end of follow-up. The long-term effects of IFX on the course of CD as reflected by the rate of surgery and hospitalisations and need for corticosteroids were also analysed. RESULTS: 10.9% of patients were primary non-responders to IFX. Sustained benefit was observed in 347 of the 547 patients (63.4%) receiving long-term treatment. In 68.3% of these, treatment with IFX was ongoing and in 31.7% IFX was stopped, with the patient being in remission. Seventy patients (12.8%) had to stop IFX due to side effects and 118 (21.6%) due to loss of response. Although the yearly drop-out rates of IFX in patients with episodic (10.7%) and scheduled treatment (7.1%) were similar, the need for hospitalisations and surgery decreased less in the episodic than in the scheduled group. Steroid discontinuation also occurred in a higher proportion of patients in the scheduled group than in the episodic group. CONCLUSIONS: In this large real-life cohort of patients with CD, long-term treatment with IFX was very efficacious to maintain improvement during a median follow-up of almost 5 years and changed disease outcome by decreasing the rate of hospitalisations and surgery.