Protective immunity induced by an inhaled SARS-CoV-2 subunit vaccine.

Targeting the site of infection is a promising strategy for improving vaccine effectivity. To date, licensed COVID-19 vaccines have been administered intramuscularly despite the fact that SARS-CoV-2 is a respiratory virus. Here, we aim to induce local protective mucosal immune responses with an inhaled subunit vaccine candidate, ISR52, based on the SARS-CoV-2 Spike S1 protein. When tested in a lethal challenge hACE2 transgenic SARS-CoV-2 mouse model, intranasal and intratracheal administration of ISR52 provided superior protection against severe infection, compared to the subcutaneous injection of the vaccine. Interestingly for a protein-based vaccine, inhaled ISR52 elicited both CD4 and CD8 T-cell Spike-specific responses that were maintained for at least 6 months in wild-type mice. Induced IgG and IgA responses cross-reacting with several SARS- CoV-2 variants of concern were detected in the lung and in serum and protected animals displayed neutralizing antibodies. Based on our results, we are developing ISR52 as a dry powder formulation for inhalation, that does not require cold-chain distribution or the use of needle administration, for evaluation in a Phase I/II clinical trial.

Upregulated expression of Notch1/4 - JAG-1/DLL-1 detected in allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is a chronic disease with high prevalence. There are currently many treatments available. However, despite an often good therapeutic response, many patients still report impairment in quality of life (QoL) during the pollen season. A skewed T helper (Th)2 polarization is a well-acknowledged pathologic feature of AR. In animal models, local notch signaling in peripheral tissue seems crucial for Th2 cell differentiation and the development of AR. However, the involvement of Notch signaling in Th2 cell differentiation and the development of AR in humans remains unknown. Hence, the present study investigated the human expression of Notch receptors on CD4+ T-cells in nasal mucosa and blood. Correspondingly Notch ligand expression was assessed on nasal epithelial cells and neutrophils. METHODOLOGY: Nasal brush and blood samples from 18 patients with pollen-induced AR and 22 healthy controls were collected outside the pollen season. Notch 1-4 and Jagged-1,2 and Delta-like ligand 1,3-4 was analyzed using flow cytometry. RESULTS: The fraction of CD4+Notch1+ and CD4+Notch4+ T-cells was higher in AR patients than in healthy control patients. Further, the expression levels of the Notch ligands JAG-1 and DLL-1 were increased in nasal epithelial cells from AR patients compared to healthy control patients. In addition, AR patients displayed higher expression of JAG-1 on neutrophils both in the nasal mucosa and in peripheral blood. CONCLUSION: The present study is the first to demonstrate increased activity in the Notch1/4 - JAG-1/DLL-1 pathways among allergic individuals. Further propagating the importance of Notch signalling in AR and blocking JAG-1 and DLL-1-induced Notch signalling by nasal epithelial cells and Neutrophils are potential targets to reduce allergic airway inflammation.

Immune-Activated B Cells Are Dominant in Prostate Cancer.

B cells are multifaceted immune cells responding robustly during immune surveillance against tumor antigens by presentation to T cells and switched immunoglobulin production. However, B cells are unstudied in prostate cancer (PCa). We used flow cytometry to analyze B-cell subpopulations in peripheral blood and lymph nodes from intermediate-high risk PCa patients. B-cell subpopulations were related to clinicopathological factors. B-cell-receptor single-cell sequencing and VDJ analysis identified clonal B-cell expansion in blood and lymph nodes. Pathological staging was pT2 in 16%, pT3a in 48%, and pT3b in 36%. Lymph node metastases occurred in 5/25 patients (20%). Compared to healthy donors, the peripheral blood CD19+ B-cell compartment was significantly decreased in PCa patients and dominated by naïve B cells. The nodal B-cell compartment had significantly increased fractions of CD19+ B cells and switched memory B cells. Plasmablasts were observed in tumor-draining sentinel lymph nodes (SNs). VDJ analysis revealed clonal expansion in lymph nodes. Thus, activated B cells are increased in SNs from PCa patients. The increased fraction of switched memory cells and plasmablasts together with the presence of clonally expanded B cells indicate tumor-specific T-cell-dependent responses from B cells, supporting an important role for B cells in the protection against tumors.

Long-lasting T-cell response to SARS-CoV-2 antigens after vaccination-a prospective cohort study of HCWs working with COVID-19 patients.

BACKGROUND: Vaccination against SARS-CoV-2 reduces the risk of hospitalisation and death, but vaccine-induced IgG antibodies against the spike protein (IgG S) decline over time. Less is known about the nature of the vaccine-induced T-cell response to SARS-CoV-2 antigens. METHODS: IgG antibodies against nucleocapsid protein (IgG N), IgG S, and T-cell response towards SARS-CoV-2 antigens were determined in samples taken between November 2020 and November 2021 from a cohort of healthcare workers at an Infectious Diseases Department. RT-PCR screening for SARS-CoV-2 was encouraged once every four weeks in addition to testing when symptomatic or identified through contact tracing. Vaccination data were collected at the end of the study. RESULTS: At inclusion, T-cell response to SARS-CoV-2 antigens was found in 10/15 (66.7%) of participants with a previous/current COVID-19 infection and in 9/54 (16.7%) of participants with no prior/current history of COVID-19 infection. All participants with complete follow-up (n = 59) received two doses of a SARS-CoV-2 vaccine during the study. All participants demonstrated detectable IgG (S) antibodies at the end of the study, in median 278 days (IQR 112) after the second vaccine dose. All but four participants displayed T-cell responses towards SARS-CoV-2 antigens. IgG S antibody levels correlated with time since the second vaccine dose. In addition, previous COVID-19 infection and the strength of the S1 T-cell response correlated with IgG S antibody levels. However, no correlation was demonstrated between the strength of the T-cell response and time since the second vaccine dose. CONCLUSION: COVID-19 vaccination induces robust T-cell responses that remain for at least nine months.

A third dose SARS­CoV­2 BNT162b2 mRNA vaccine results in improved immune response in hemodialysis patients.

Background: The hemodialysis (HD) population has been a vulnerable group during the coronavirus disease 2019 (COVID-19) pandemic. Advanced chronic kidney disease with uremia is associated with weaker immune response to infections and an attenuated response to vaccines. The aim of this study was to study the humoral and cellular response to the second and third doses of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS­CoV­2) BNT162b2 mRNA vaccine in HD patients and to follow the response over time. Methods: The patients received their first two vaccine doses from 28 December 2020 within a 4-week interval and the third dose in September 2021 and were followed-up for humoral and cellular immune response at 1) 7-15 weeks and 2) 6-8 months after dose two (no t-cell reactivity measured), and 3) 3 weeks and 4) 3 months after dose three. Fifty patients were initially enrolled, and 40 patients were followed during the entire study. Levels of COVID-19 (SARS-CoV-2) IgG antibody against the Spike antigen (anti-S) and T-cell reactivity testing against the Spike protein using Enzyme-Linked ImmunoSpot (ELISPOT) technology were evaluated. Results: IgG antibodies to anti-S were detected in 35 (88%) of the 40 patients 7-15 weeks after vaccine dose two, 31 (78%) were positive, and 4 (10%) borderline. The median anti-S titer was 606 Abbott Units/milliliter (AU/mL) (interquartile range [IQR] 134-1,712). Three months after the third dose, anti-S was detected in 38 (95%) of 40 patients (P < 0.01 compared to after dose two), and the median anti-S titer was 9,910 AU/mL (IQR 2,325-26,975). Cellular reactivity was detected in 22 (55%), 34 (85%), and 28 (71%) of the 40 patients, and the median T-cell response was 9.5 (IQR 3.5-80), 51.5 (14.8-132), and 19.5 (8.8-54.2) units, respectively, for 6-8 months after dose two, 3 weeks, and 3 months after dose three. Conclusions: Our data show that a third dose of SARS­CoV­2 BNT162b2 mRNA vaccine gives a robust and improved immunological response in HD patients, but a few patients did not develop any anti-S response during the entire study, indicating the importance to monitor the vaccine response since those who do not respond could now be given monoclonal antibodies if they contract a COVID-19 infection or in the future antivirals.

Monitoring of the SARS-CoV-2 Omicron BA.1/BA.2 lineage transition in the Swedish population reveals increased viral RNA levels in BA.2 cases.

BACKGROUND: Throughout the SARS-CoV-2 pandemic, multiple waves of variants of concern have swept across populations, leading to a chain of new and yet more contagious variants dominating COVID-19 cases. Here, we tracked the remarkably rapid shift from Omicron BA.1 to BA.2 sublineage dominance in the Swedish population in early 2022 at a day-by-day basis. METHODS: Using a custom SARS-CoV-2 Omicron BA.1 lineage-typing RT-PCR assay, we analyzed 174,933 clinical upper airway samples collected during January to March 2022. FINDINGS: Our study demonstrates the feasibility and reliability of parallel lineage assignment of select variants at population scale, tracking the dominant sublineage transition from BA.1 to BA.2 at day-to-day resolution and uncovering nearly 2-fold higher levels of viral RNA in cases infected with Omicron BA.2 relative to BA.1. CONCLUSIONS: Our data provide unique insights into the Omicron BA.1 to BA.2 transition that occurred in Sweden during early 2022, and later, across the world. This may help to understand the increased transmissibility of the BA.2 variant.

Genetic variability in exon 1 of the glucocorticoid receptor gene NR3C1 is associated with postoperative complications.

Patients undergoing major surgery experience postoperative inflammation, which may contribute to postoperative morbidity. Endogenous glucocorticoids (GCs) are an essential part of the stress response, but this response varies between individuals, which may in turn affect clinical outcome and specifically postoperative inflammation. Exon 1 of the NR3C1 gene, encoding the GC receptor (GR), contains an established region of differential regulation. DNA methylation patterns in this region have been found to differ between individuals. The present study investigated the methylation status and genotype in the cytosine­phosphate­guanine (CpG) island in exon 1 of NR3C1 in 24 patients [Median age 65.5 (range 42­81) years, 11 male, 13 female] who underwent major abdominal (12 pancreatic, 12 hepatic) surgery and explored its association with postoperative complications. DNA was extracted from peripheral blood leukocytes and underwent targeted bisulfite sequencing of the CpG island. Complications were graded according to the Clavien­Dindo classification and 14 out of 24 patients had postoperative complications. Multifactorial and partial least square analyses were used to analyse the data. A homogenous demethylated pattern was observed in all patients and no single CpG methylation was associated with postoperative complications. Four SNPs were significantly associated with higher Clavien­Dindo scores. Genetic variability in the chromosome 5:143,402,505­143,405,805 region of exon 1 of the GR gene NR3C1, but not DNA methylation, was associated with more severe postoperative complications in patients having major abdominal surgery. These results indicated that the patients' response to GCs may be of clinical importance for inflammatory conditions.

High-dose pollen intralymphatic immunotherapy: Two RDBPC trials question the benefit of dose increase.

BACKGROUND: The same dosing schedule, 1000 SQ-U times three, with one-month intervals, have been evaluated in most trials of intralymphatic immunotherapy (ILIT) for the treatment of allergic rhinitis (AR). The present studies evaluated if a dose escalation in ILIT can enhance the clinical and immunological effects, without compromising safety. METHODS: Two randomized double-blind placebo-controlled trials of ILIT for grass pollen-induced AR were performed. The first included 29 patients that had recently ended 3 years of SCIT and the second contained 39 not previously vaccinated patients. An up-dosage of 1000-3000-10,000 (5000 + 5000 with 30 minutes apart) SQ-U with 1 month in between was evaluated. RESULTS: Doses up to 10,000 SQ-U were safe after recent SCIT. The combined symptom-medication scores (CSMS) were reduced by 31% and the grass-specific IgG4 levels in blood were doubled. In ILIT de novo, the two first patients that received active treatment developed serious adverse reactions at 5000 SQ-U. A modified up-dosing schedule; 1000-3000-3000 SQ-U appeared to be safe but failed to improve the CSMS. Flow cytometry analyses showed increased activation of lymph node-derived dendritic but not T cells. Quality of life and nasal provocation response did not improve in any study. CONCLUSION: Intralymphatic immunotherapy in high doses after SCIT appears to further reduce grass pollen-induced seasonal symptoms and may be considered as an add-on treatment for patients that do not reach full symptom control after SCIT. Up-dosing schedules de novo with three monthly injections that exceeds 3000 SQ-U should be avoided.

Humoral and cellular response to SARS-CoV-2 BNT162b2 mRNA vaccine in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients have an increased risk of acquiring infections due to many health care contacts and may, in addition, have a suboptimal response to vaccination and a high mortality from Covid-19 infection. METHODS: In 50 HD patients (mean age 69.4 years, 62% men) administration of SARS-CoV-2BNT162b2 mRNA vaccine began in Dec 2020 and the immune response was evaluated 7-15 weeks after the last dose. Levels of Covid-19 (SARS-CoV-2) IgG antibody against the nucleocapsid antigen (anti-N) and the Spike antigen (anti-S) and T-cell reactivity testing against the Spike protein using ELISPOT technology were evaluated. RESULTS: Out of 50 patients, anti-S IgG antibodies indicating a vaccine effect or previous Covid-19 infection, were detected in 37 (74%), 5 (10%) had a borderline response and 8 (16%) were negative after two doses of vaccine. T-cell responses were detected in 29 (58%). Of the 37 patients with anti-S antibodies, 25 (68%) had a measurable T-cell response. 2 (40%) out of 5 patients with borderline anti-S and 2 (25%) without anti-S had a concomitant T-cell response. Twenty-seven (54%) had both an antibody and T-cell response. IgG antibodies to anti-N indicating a previous Covid-19 disease were detected in 7 (14%) patients. CONCLUSIONS: Most HD patients develop a B- and/or T-cell response after vaccination against Covid-19 but approx. 20% had a limited immunological response. T-cell reactivity against Covid-19 was only present in a few of the anti-S antibody negative patients.

Proteomic Profiling of Tissue Exosomes Indicates Continuous Release of Malignant Exosomes in Urinary Bladder Cancer Patients, Even with Pathologically Undetectable Tumour.

Invasive urothelial bladder cancer (UBC) has high recurrence rates even after radical cystectomy (RC). Exosomes are membrane-bound nanovesicles, which have been shown to contribute to carcinogenesis and metastasis. We previously showed that urinary exosomes display a malignant profile in UBC patients despite the absence of detectable tumour. Here, we investigated exosomes from sampling sites close to or distant from the former tumour, aiming to understand the effect of the tumour on the local milieu. Ten patients scheduled for cystectomy after transurethral bladder resection (TUR-B), without remaining detectable tumour, were included. Exosomes were isolated from tissue explants of both the previous tumour site and distant bladder tissue. Proteins were quantified by mass spectrometry in seven patients. Exosomes from the previous tumour site were enriched in inflammatory but not cancer-related pathways compared to distant tissue. However, the 69 most abundant proteins in tissue-derived exosomes regardless of site, 20 of which were also found in urinary exosomes from our previous study, were enriched for cancer-related metabolic pathways and associated with poor prognosis in an external mRNA dataset. The enrichment of cancer-related pathways in the most abundant proteins, regardless of sampling site, confirms our hypothesis that despite the absence of detectable tumour, the entire bladder releases exosomes that contribute to metastasis and highlights the need for early RC.

Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.

BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

The intratumoral CXCR3 chemokine system is predictive of chemotherapy response in human bladder cancer.

Chemotherapy has direct toxic effects on cancer cells; however, long-term cancer control and complete remission are likely to involve CD8+ T cell immune responses. To study the role of CD8+ T cell infiltration in the success of chemotherapy, we examined patients with muscle invasive bladder cancer (MIBC) who were categorized on the basis of the response to neoadjuvant chemotherapy (NAC). We identified the intratumoral CXCR3 chemokine system (ligands and receptor splice variants) as a critical component for tumor eradication upon NAC in MIBC. Through characterization of CD8+ T cells, we found that stem-like T cell subpopulations with abundant CXCR3alt, a variant form of the CXCL11 receptor, responded to CXCL11 in culture as demonstrated by migration and enhanced effector function. In tumor biopsies of patients with MIBC accessed before treatment, CXCL11 abundance correlated with high numbers of tumor-infiltrating T cells and response to NAC. The presence of CXCR3alt and CXCL11 was associated with improved overall survival in MIBC. Evaluation of both CXCR3alt and CXCL11 enabled discrimination between responder and nonresponder patients with MIBC before treatment. We validated the prognostic role of the CXCR3-CXCL11 chemokine system in an independent cohort of chemotherapy-treated and chemotherapy-naïve patients with MIBC from data in TCGA. In summary, our data revealed stimulatory activity of the CXCR3alt-CXCL11 chemokine system on CD8+ T cells that is predictive of chemotherapy responsiveness in MIBC. This may offer immunotherapeutic options for targeted activation of intratumoral stem-like T cells in solid tumors.

Immune responses against autologous tumor and human papilloma virus in lymph nodes from patients with penile cancer.

PURPOSE: Nearly half of penile cancers are related to human papillomavirus (HPV) infection. Investigations of tumor- and HPV-specific T cell reactivity in regional lymph nodes (LNs) from patients with penile cancer are warranted. MATERIALS AND METHODS: In this study, single-cell suspensions from LNs and peripheral blood from 11 patients with penile cancer were stained with antibodies for lymphocyte markers and analyzed by fluorescence-activated cell sorting (FACS). DNA was extracted from the tumor tissue and HPV status was investigated by PCR. RESULTS: T-cell reactivity against autologous tumor-extract and against the HPV-vaccine Gardasil® was tested by flow-cytometric assay of specific cell-mediated immune response in activated whole blood (FASCIA). CD4+/CD8+ ratios were significantly lower in HPV positive LNs (p<0.05). Immune responses to tumor extract assessed by blast transformation and expansion in vitro, of either CD4+ or CD8+ T-cells, were found in 9 of 13 LNs (69%). 5 of 6 tested patients demonstrated T cell recognition of tumor-associated antigen(s). In HPV-positive patients, dose-dependent T cell responses against L1 (late) HPV proteins (Gardasil vaccine) were demonstrated. CONCLUSIONS: LN-derived T cells from patients with penile cancer recognize tumor antigen(s) and in HPV-positive cases, there is a response against L1 (late) HPV proteins, being constituents of the Gardasil vaccine.

Hodgkin Lymphoma Monozygotic Triplets Reveal Divergences in DNA Methylation Signatures.

We studied DNA methylation profiles in four different cell populations from a unique constellation of monozygotic triplets in whom two had developed Hodgkin Lymphoma (HL). We detected shared differences in DNA methylation signatures when comparing the two HL-affected triplets with the non-affected triplet. The differences were observed in naïve B-cells and marginal zone-like B-cells. DNA methylation differences were also detected when comparing each of the HL-affected triplets against each other. Even though we cannot determine whether treatment and/or disease triggered the observed differences, we believe our data are important on behalf of forthcoming studies, and that it might provide important clues for a better understanding of HL pathogenesis.

Activation of T helper cells in sentinel node predicts poor prognosis in oral squamous cell carcinoma.

Recurrence in oral squamous cell carcinoma (OSCC) significantly reduces overall survival. Improved understanding of the host's immune status in head and neck cancer may facilitate identification of patients at higher risk of recurrence and improve patients' selection for ongoing clinical trials assessing the effectiveness of immune checkpoint inhibitors (CPI). We aimed to investigate Sentinel Node-derived T-cells and their impact on survival. We enrolled prospectively 28 OSCC patients treated at Karolinska University Hospital, Stockholm, Sweden with primary tumour excision and elective neck dissection. On top of the standard treatment, the enrolled patients underwent sentinel node procedure. T cells derived from Sentinel nodes, non-sentinel nodes, primary tumour and PBMC were analyzed in flow cytometry. Patients who developed recurrence proved to have significantly lower level of CD4+ CD69+ in their sentinel node (31.38 ± 6.019% vs. 43.44 ± 15.33%, p = 0.0103) and significantly higher level of CD8+ CD HLA-DR+ (38.95 ± 9.479% vs. 24.58 ± 11.36%, p = 0.0116) compared to disease-free individuals. Survival analysis of studied population revealed that patients with low proportion of CD4+ CD69+ had significantly decreased disease-free survival (DFS) of 19.7 months (95% CI 12.6-26.9) compared with 42.6 months (95% CI 40.1-45.1) in those with high CD4+ CD69+ proportion in their Sentinel Nodes (log-rank test, p = 0.033). Our findings demonstrate that characterization of T-cell activation in Sentinel Node serves as a complementary prognostic marker. Flow cytometry of Sentinel Node may be useful in both patients' surveillance and selection for ongoing CPI clinical trials in head and neck cancer.

IL-16 processing in sentinel node regulatory T cells is a factor in bladder cancer immunity.

In the effort of developing new immunotherapies, the sentinel node (SN) has proven a promising source from which to harness an effective antitumour T cell response. However, tumour immune escape, a process in which regulatory T cells (Tregs) play a central role, remains a major limiting factor. Therefore, there is a clear need to increase the knowledge of Treg function and signalling in sentinel nodes. Here, we set out to explore whether the proteome in SN-resident T cells is altered by the tumour and to identify key proteins in SN T cell signalling, focusing on Tregs. Five patients with muscle-invasive urothelial bladder cancer were prospectively included. Mass spectrometry was performed on two patients, with validation and functional studies being performed on three additional patients and four healthy donors. At cystectomy, SN, non-SN lymph nodes and peripheral blood samples were collected from the patients and T cell subsets isolated through flow cytometry before downstream experiments. Proteomic analysis indicated that growth and immune signalling pathways are upregulated in SN-resident Tregs. Furthermore, centrality analysis identified the cytokine IL-16 to be central in the SN-Treg signalling network. We show that tumour-released factors, through activating caspase-3, increase Treg IL-16 processing into bioactive forms, reinforcing Treg suppressive capacity. In conclusion, we provide evidence that Tregs exposed to secreted factors from bladder tumours show increased immune and growth signalling and altered IL-16 processing which translates to enhanced Treg suppressive function, indicating altered IL-16 signalling as a novel tumour immune escape mechanism.

Urinary Exosomes from Bladder Cancer Patients Show a Residual Cancer Phenotype despite Complete Pathological Downstaging.

Invasive urinary bladder cancer shows high recurrence rates after cystectomy even with apparent complete downstaging at cystectomy. Exosomes are nano-sized vesicles important in cell-cell communication, which have been hypothesized to contribute to cancer dissemination and recurrence. The aim of this study was to investigate if pro-carcinogenic exosomes could be detected in urine from histologically downstaged bladder cancer patients. 13 Patients were included in this study. Paired ureter and urine samples from nine patients underwent mass spectrometry, while samples from the remaining patients were used for exosome characterization. At cystectomy, exosomes were isolated from bladder and ureter urine, whereafter quantitative proteome profiling was performed. Urinary exosomes clustered based on whether they came from the bladder, with tumour contact, or the ureters, without tumour contact, even though all came from completely downstaged patients. Proteins overexpressed in exosomes derived from bladder urine contained several oncogenes and were mainly associated with tumour metabolism pathways. Although patients were histologically tumour-free at cystectomy, the bladder urine contained exosomes with a carcinogenic metabolic profile. This suggests a continuous release of exosomes from the bladder, which may promote recurrence at distant sites through metabolic rewiring, even after apparent complete downstaging. These exosomes, coming from either undetected cancer cells or partly transformed cells, are likely to increase the risk of metastasis and encourages cystectomy even in completely downstaged patients.

Blood transfusions during neoadjuvant chemotherapy for muscle-invasive urinary bladder cancer may have a negative impact on overall survival.

Objective: To evaluate the extent and plausible effects of blood transfusions given during cisplatin-based neoadjuvant chemotherapy (NAC) on overall survival in patients with muscle-invasive urothelial bladder cancer (MIBC) undergoing NAC and radical cystectomy (RC).Background: Several studies have demonstrated a decreased survival for MIBC patients receiving allogenic peri- and postoperative blood transfusions in conjunction with RC. No studies have previously investigated the effects of blood transfusions during NAC.Materials and methods: 120 patients with MIBC (cT2-T4aN0M0) undergoing NAC and RC between 2008 and 2014 at four Swedish cystectomy centers were retrospectively evaluated. Clinicopathological data were obtained, including data of allogenic blood administration. Survival data was analyzed by Kaplan-Meier plotting and Cox regression.Results: One third of the cohort received blood transfusions during the period of NAC. In univariate analysis, blood transfusions during NAC, nodal stage and advanced tumor stage (pT >2) were negative prognostic factors for survival. In multivariate analysis, only pNx and pT >2 remained significant negative prognostic factors. In a subgroup analysis consisting of patients with localized tumors without dissemination (n = 96), patients that received transfusions during NAC showed an 18.5% absolute risk increase of death at five years of observation, although without statistical significance (p = .197).Conclusions: This is the first time that the extent and plausible effects of allogenic blood transfusions during NAC is examined in MIBC. Data suggest that there may be an association between blood transfusion and poor pathological and oncological outcome. Firm conclusions are difficult to draw due to few study participants and the retrospective nature of the study.

An intronic deletion in megakaryoblastic leukemia 1 is associated with hyperproliferation of B cells in triplets with Hodgkin lymphoma.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Tumour-associated B cells in urothelial urinary bladder cancer.

Tumour infiltrating B cells and CD38+ plasma cells have been correlated with survival in different malignancies but their role in urinary bladder cancer is unclear. IL-10 is a multifunctional cytokine with both anti-inflammatory and immunostimulatory properties, that can be released by regulatory B cells (Bregs). We have stained paraffin-embedded tumour sections from 31 patients with invasive urothelial urinary bladder cancer with respect to CD20+ B cells, CD38+ cells, IL-10-expressing cells, IgG, C1q and C3a and analysed the impact of these markers on survival. Interestingly, we observe tumour-associated CD20+ B cells forming follicle-like structures in tumours of some patients. We demonstrate that follicle-like structures, tumour-associated CD38+ cells, IL-10 produced by non-B cells, tumour infiltrating IgG and activation of the complement system, may associate to longer survival of urinary bladder cancer patients. IL-10 expression by tumour-associated Bregs may instead negatively affect prognosis. More research is needed to fully understand the role of B cells and IL-10 in urinary bladder cancer.