Atypical B cells promote cancer progression and poor response to Bacillus Calmette-Guérin in non-muscle invasive bladder cancer.

Poor response to Bacillus Calmette-Guérin (BCG) immunotherapy remains a major barrier in the management of patients with non-muscle invasive bladder cancer (NMIBC). Multiple factors are associated with poor outcomes, including biological aging and female sex. More recently, it has emerged that a B-cell infiltrated pre-treatment immune microenvironment of NMIBC tumors can influence the response to intra-vesically administered BCG. The mechanisms underlying the roles of B cells in NMIBC are poorly understood. Here, we show that B-cell dominant tertiary lymphoid structures (TLSs), a hallmark feature of the chronic mucosal immune response, are abundant and located close to the epithelial compartment in pre-treatment tumors from BCG non-responders. Digital spatial proteomic profiling of whole tumor sections from male and female patients with NMIBC who underwent treatment with intravesical BCG, revealed higher expression of immune exhaustion-associated proteins within the tumor-adjacent TLSs in both responders and non-responders. Chronic local inflammation, induced by the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen, led to TLS formation with recruitment and differentiation of the immunosuppressive atypical B-cell (ABC) subset within the bladder microenvironment, predominantly in aging female mice compared to their male counterparts. Depletion of ABCs simultaneous to BCG treatment delayed cancer progression in female mice. Our findings provide evidence indicating a role for ABCs in BCG response and will inform future development of therapies targeting the B cell-exhaustion axis.

Quantitative Nuclear Grading: An Objective, Artificial Intelligence-Facilitated Foundation for Grading Noninvasive Papillary Urothelial Carcinoma.

In nonmuscle invasive bladder cancer, grade drives important treatment and management decisions. However, grading is complex and qualitative, and it has considerable interobserver and intraobserver variability. Previous literature showed that nuclear features quantitatively differ between bladder cancer grades, but these studies were limited in size and scope. In this study, we aimed to measure morphometric features relevant to grading criteria and build simplified classification models that objectively distinguish between the grades of noninvasive papillary urothelial carcinoma (NPUC). We analyzed 516 low-grade and 125 high-grade 1.0-mm diameter image samples from a cohort of 371 NPUC cases. All images underwent World Health Organization/International Society of Urological Pathology 2004 consensus pathologist grading at our institution that was subsequently validated by expert genitourinary pathologists from 2 additional institutions. Automated software segmented the tissue regions and measured the nuclear features of size, shape, and mitotic rate for millions of nuclei. Then, we analyzed differences between grades and constructed classification models, which had accuracies up to 88% and areas under the curve as high as 0.94. Variation in the nuclear area was the best univariate discriminator and was prioritized, along with the mitotic index, in the top-performing classifiers. Adding shape-related variables improved accuracy further. These findings indicate that nuclear morphometry and automated mitotic figure counts can be used to objectively differentiate between grades of NPUC. Future efforts will adapt the workflow to whole slides and tune grading thresholds to best reflect time to recurrence and progression. Defining these essential quantitative elements of grading has the potential to revolutionize pathologic assessment and provide a starting point from which to improve the prognostic utility of grade.

Sex differences in the aging murine urinary bladder and influence on the tumor immune microenvironment of a carcinogen-induced model of bladder cancer.

Sex and age associated differences in the tumor immune microenvironment of non-muscle invasive bladder (NMIBC) cancer and associated clinical outcomes are emerging indicators of treatment outcomes. The incidence of urothelial carcinoma of the bladder is four times higher in males than females; however, females tend to present with a more aggressive disease, a poorer response to immunotherapy and suffer worse clinical outcomes. Recent findings have demonstrated sex differences in the tumor immune microenvironment of non-muscle invasive and muscle invasive bladder cancer and associated clinical outcomes. However, a significant gap in knowledge remains with respect to the current pre-clinical modeling approaches to more precisely recapitulate these differences towards improved therapeutic design. Given the similarities in mucosal immune physiology between humans and mice, we evaluated the sex and age-related immune alterations in healthy murine bladders. Bulk-RNA sequencing and multiplex immunofluorescence-based spatial immune profiling of healthy murine bladders from male and female mice of age groups spanning young to old showed a highly altered immune landscape that exhibited sex and age associated differences, particularly in the context of B cell mediated responses. Spatial profiling of healthy bladders, using markers specific to macrophages, T cells, B cells, activated dendritic cells, high endothelial venules, myeloid cells and the PD-L1 immune checkpoint showed sex and age associated differences. Bladders from healthy older female mice also showed a higher presence of tertiary lymphoid structures (TLSs) compared to both young female and male equivalents. Spatial immune profiling of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen exposed male and female bladders from young and old mice revealed a similar frequency of TLS formation, sex differences in the bladder immune microenvironment and, age associated differences in latency of tumor induction. These findings support the incorporation of sex and age as factors in pre-clinical modeling of bladder cancer and will potentially advance the field of immunotherapeutic drug development to improve clinical outcomes.

A miR-375/YAP axis regulates neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

Lung carcinoids are variably aggressive and mechanistically understudied neuroendocrine neoplasms (NENs). Here, we identified and elucidated the function of a miR-375/yes-associated protein (YAP) axis in lung carcinoid (H727) cells. miR-375 and YAP are respectively high and low expressed in wild-type H727 cells. Following lentiviral CRISPR/Cas9-mediated miR-375 depletion, we identified distinct transcriptomic changes including dramatic YAP upregulation. We also observed a significant decrease in neuroendocrine differentiation and substantial reductions in cell proliferation, transformation, and tumor growth in cell culture and xenograft mouse disease models. Similarly, YAP overexpression resulted in distinct and partially overlapping transcriptomic changes, phenocopying the effects of miR-375 depletion in the same models as above. Transient YAP knockdown in miR-375-depleted cells reversed the effects of miR-375 on neuroendocrine differentiation and cell proliferation. Pathways analysis and confirmatory real-time PCR studies of shared dysregulated target genes indicate that this axis controls neuroendocrine related functions such as neural differentiation, exocytosis, and secretion. Taken together, we provide compelling evidence that a miR-375/YAP axis is a critical mediator of neuroendocrine differentiation and tumorigenesis in lung carcinoid cells.

Quantitative Analysis of SARS-CoV-2 Antibody Status between Patients with Cancer and Healthy Individuals with Extended Vaccination Dosing Intervals in Canada.

(1) Background: To date, data addressing the antibody response of cancer patients to SARS-CoV-2 vaccines are limited. To our knowledge, this is the first report to evaluate humoral immunity. responses in Canadian cancer patients. (2) Methods: 116 cancer patients and 35 healthy participants were enrolled in this cross-sectional study. The interval between the first and second doses were closely matched during analysis. IgG antibodies against the SARS-CoV-2 spike receptor-binding domain were determined using an enzyme-linked immunosorbent assay (ELISA). (3) Results: Following two doses of SARS-CoV-2 vaccine (including BNT162b2, AZD1222, and mRNA-1273), the mean serum anti-spike protein antibody level was 382.4 BAU/mL (binding antibody unit, SD ± 9.4) in the control group, 265.8 BAU/mL (±145.7) in solid cancer patients, and 168.2 BAU/mL (±172.9) in hematological cancer patients. Observed differences were significantly lower in both solid and hematological groups when comparing to the control group (p ≤ 0.0001). In solid cancer group, patients with cytotoxic chemotherapy demonstrated significantly lower antibody levels (p < 0.01), whereas the rest of the patients showed similar antibody levels as the healthy control. Antibody levels were lower in those on treatment than those off treatment in patients with hematological malignancies (p < 0.0001) but not for those with solid cancers (p = 0.4553). (4) Conclusions: After two doses of the SARS-CoV-2 vaccination, patients with solid and hematological malignancies demonstrated impaired serological responses. This was particularly prominent if there was cytotoxic chemotherapy or systemic therapy in solid and hematological cancer, respectively.

Assessment of tissue oxygenation of periodontal inflammation in patients with coronary artery diseases using optical spectroscopy.

BACKGROUND: We have recently developed a non-invasive periodontal diagnostic tool that was validated in periodontitis patients without systemic disorders like coronary artery disease (CAD). The purpose of present study is to verify whether this optical instrument can also be used in periodontitis patients with CAD. METHODS: A total of 62 periodontitis patients with CAD were recruited along with a control group consisting of 59 age and gender matched periodontitis volunteers without systemic disorders. Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed and evaluated from the two groups. A modified Beer-Lambert unmixing model that incorporates a nonparametric scattering loss function was used to determine the relative contribution of deoxygenated hemoglobin (Hb) and oxygenated hemoglobin (HbO2) to the overall spectrum. The balance between tissue oxygen delivery and utilization in periodontal tissues was then assessed. RESULTS: Tissue oxygen saturation was significantly decreased in the periodontitis sites (p < 0.01), compared to the healthy sites in those individuals with CAD. There was a trend towards increased concentration of Hb and decreased concentration of HbO2 from healthy to diseased sites, without statistical significance (p > 0.05). No statistical differences were found in tissue oxygen saturation between the CAD and control groups either in periodontal healthy or inflammatory sites. CONCLUSION: This study supports the hypothesis that optical spectroscopy can determine the periodontal inflammation in patients with certain systemic disorders like CAD. And the overall periodontal oxygenation profiles in CAD patients resemble those in non-CAD individuals either in healthy or inflammatory sites.

Assessment of tissue oxygenation of periodontal inflammation in smokers using optical spectroscopy.

BACKGROUND: We have recently developed a periodontal diagnostic tool that was validated in non-smokers with periodontitis. Tobacco smoking is a recognized risk factor for periodontal diseases that can mask gingival bleeding and lead to a false negative diagnosis. Therefore, the purpose of current study is to further validate this instrument in smokers with periodontal diseases. METHODS: Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed and evaluated from healthy (n = 108), gingivitis (n = 100), and periodontitis (n = 79) sites of 54 systemically healthy smokers. A modified Beer-Lambert unmixing model that incorporates a non-parametric scattering loss function was used to determine the relative contribution of deoxygenated haemoglobin (Hb) and oxygenated haemoglobin (HbO2 ) to the overall spectrum. The balance between tissue oxygen delivery and utilization in periodontal tissues was then assessed. RESULTS: Tissue oxygen saturation was significantly decreased in the gingivitis (p = 0.016) and periodontitis (p = 0.007) sites, compared to the healthy sites. There was a trend towards increased concentration of Hb and decreased concentration of HbO2 from healthy to diseased sites, without statistical significance (p > 0.05). CONCLUSIONS: Optical spectroscopy can determine tissue oxygenation profiles of healthy and diseased sites in smokers. The spectral profile of periodontal sites in smokers generally resembles those from non-smoking patients.

The influence of nicotine on granulocytic differentiation - inhibition of the oxidative burst and bacterial killing and increased matrix metalloproteinase-9 release.

BACKGROUND: Neutrophils leave the bone marrow as terminally differentiated cells, yet little is known of the influence of nicotine or other tobacco smoke components on neutrophil differentiation. Therefore, promyelocytic HL-60 cells were differentiated into neutrophils using dimethylsulfoxide in the presence and absence of nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine). Differentiation was evaluated over 5 days by monitoring terminal differentiation markers (CD11b expression and formazan deposition); cell viability, growth phase, kinetics, and apoptosis; assessing cellular morphology and ultrastructure; and conformational changes to major cellular components. Key neutrophil effector functions (oxidative burst, bacterial killing, matrix metalloproteinase release) were also examined. RESULTS: Nicotine increased the percentage of cells in late differentiation phases (metamyelocytes, banded neutrophils and segmented neutrophils) compared to DMSO alone (p < 0.05), but did not affect any other marker of neutrophil differentiation examined. However, nicotine exposure during differentiation suppressed the oxidative burst in HL-60 cells (p < 0.001); inhibited bacterial killing (p < 0.01); and increased the LPS-induced release of MMP-9, but not MMP-2 (p < 0.05). These phenomena may be alpha-7-acetylcholine nicotinic receptor-dependent. Furthermore, smokers exhibited an increased MMP-9 burden compared to non-smokers in vivo (p < 0.05). CONCLUSION: These findings may partially explain the known increase in susceptibility to bacterial infection and neutrophil-associated destructive inflammatory diseases in individuals chronically exposed to nicotine.

Biomolecular characterisation of leucocytes by infrared spectroscopy.

Over the last 15 years, infrared (IR) spectroscopy has developed into a novel and powerful biomedical tool that has multiple applications in the field of haematology. By revealing subtle alterations in both the conformation and concentration of key macromolecules, such as DNA, protein and lipids, IR spectroscopy has been employed to investigate multiple aspects of leucocyte physiology. IR spectroscopy has been used, for example, to diagnose and prognose leukaemia; to characterise differentiation and apoptotic processes; to predict drug sensitivity and resistance in leukaemic patients undergoing chemotherapy; to monitor the response of leucocytes to chemotherapy and to perform human leucocyte antigen matching for bone marrow transplant patients. Such studies have provided insight into pathogenic mechanisms underlying specific leucocyte disorders, especially leukaemia. While it is likely to be some considerable time before IR spectroscopy is sufficiently developed to displace the established technologies, IR spectroscopy has the potential to become a valuable analytic tool in basic and clinical haematology.