Amphiregulin from regulatory T cells promotes liver fibrosis and insulin resistance in non-alcoholic steatohepatitis.

Production of amphiregulin (Areg) by regulatory T (Treg) cells promotes repair after acute tissue injury. Here, we examined the function of Treg cells in non-alcoholic steatohepatitis (NASH), a setting of chronic liver injury. Areg-producing Treg cells were enriched in the livers of mice and humans with NASH. Deletion of Areg in Treg cells, but not in myeloid cells, reduced NASH-induced liver fibrosis. Chronic liver damage induced transcriptional changes associated with Treg cell activation. Mechanistically, Treg cell-derived Areg activated pro-fibrotic transcriptional programs in hepatic stellate cells via epidermal growth factor receptor (EGFR) signaling. Deletion of Areg in Treg cells protected mice from NASH-dependent glucose intolerance, which also was dependent on EGFR signaling on hepatic stellate cells. Areg from Treg cells promoted hepatocyte gluconeogenesis through hepatocyte detection of hepatic stellate cell-derived interleukin-6. Our findings reveal a maladaptive role for Treg cell-mediated tissue repair functions in chronic liver disease and link liver damage to NASH-dependent glucose intolerance.

European Association of Urology Guidelines on Muscle-invasive and Metastatic Bladder Cancer: Summary of the 2023 Guidelines.

CONTEXT: We present an overview of the updated 2023 European Association of Urology (EAU) guidelines for muscle-invasive and metastatic bladder cancer (MMIBC). OBJECTIVE: To provide practical evidence-based recommendations and consensus statements on the clinical management of MMIBC with a focus on diagnosis and treatment. EVIDENCE ACQUISITION: A broad and comprehensive scoping exercise covering all areas of the MMIBC guidelines has been performed annually since 2017. Searches cover the Medline, EMBASE, and Cochrane Libraries databases for yearly guideline updates. A level of evidence and strength of recommendation are assigned. The evidence cutoff date for the 2023 MIBC guidelines was May 4, 2022. EVIDENCE SYNTHESIS: Patients should be counselled regarding risk factors for bladder cancer. Pathologists should describe tumour and lymph nodes in detail, including the presence of histological subtypes. The importance of the presence or absence of urothelial carcinoma (UC) in the prostatic urethra is emphasised. Magnetic resonance imaging (MRI) of the bladder is superior to computed tomography (CT) for disease staging, specifically in differentiating T1 from T2 disease, and may lead to a change in treatment approach in patients at high risk of an invasive tumour. Imaging of the upper urinary tract, lymph nodes, and distant metastasis is performed with CT or MRI; the additional value of flurodeoxyglucose positron emission tomography/CT still needs to be determined. Frail and comorbid patients should be evaluated by a multidisciplinary team. Postoperative histology remains the most important prognostic variable, while circulating tumour DNA appears to be an interesting predictive marker. Neoadjuvant systemic therapy remains cisplatin-based. In motivated and selected women and men, sexual organ-preserving cystectomy results in better functional outcomes without compromising oncological outcomes. Robotic and open cystectomy have comparable outcomes and should be combined with (extended) lymph node dissection. The diversion type is an individual choice after taking patient and tumour characteristics into account. Radical cystectomy remains a highly complex procedure with considerable morbidity and risk of mortality, although lower rates are observed for higher hospital volumes (>20 cases/yr). With proper patient selection, trimodal therapy (chemoradiation) has comparable outcomes to radical cystectomy. Adjuvant chemotherapy after surgery improves disease-specific survival and overall survival (OS) in patients with high-risk disease who did not receive neoadjuvant treatment, and is strongly recommended. There is a weak recommendation for adjuvant nivolumab, as OS data are not yet available. Health-related quality of life should be assessed using validated questionnaires at baseline and after treatment. Surveillance is needed to monitor for recurrent cancer and functional outcomes. Recurrences detected on follow-up seem to have better prognosis than symptomatic recurrences. CONCLUSIONS: This summary of the 2023 EAU guidelines provides updated information on the diagnosis and treatment of MMIBC for incorporation into clinical practice. PATIENT SUMMARY: The European Association of Urology guidelines panel on muscle-invasive and metastatic bladder cancer has released an updated version of the guideline containing information on diagnosis and treatment of this disease. Recommendations are based on studies published up to May 4, 2022. Surgical removal of the bladder and bladder preservation are discussed, as well as updates on the use of chemotherapy and immunotherapy in localised and metastatic disease.

Immune checkpoints are predominantly co-expressed by clonally expanded CD4+FoxP3+ intratumoral T-cells in primary human cancers.

BACKGROUND: In addition to anti-PD(L)1, anti-CTLA-4 and anti-LAG-3, novel immune checkpoint proteins (ICP)-targeted antibodies have recently failed to demonstrate significant efficacy in clinical trials. In these trials, patients were enrolled without screening for drug target expression. Although these novel ICP-targeted antibodies were expected to stimulate anti-tumor CD8 + T-cells, the rationale for their target expression in human tumors relied on pre-clinical IHC stainings and transcriptomic data, which are poorly sensitive and specific techniques for assessing membrane protein expression on immune cell subsets. Our aim was to describe ICP expression on intratumoral T-cells from primary solid tumors to better design upcoming neoadjuvant cancer immunotherapy trials. METHODS: We prospectively performed multiparameter flow cytometry and single-cell RNA sequencing (scRNA-Seq) paired with TCR sequencing on freshly resected human primary tumors of various histological types to precisely determine ICP expression levels within T-cell subsets. RESULTS: Within a given tumor type, we found high inter-individual variability for tumor infiltrating CD45 + cells and for T-cells subsets. The proportions of CD8+ T-cells (~ 40%), CD4+ FoxP3- T-cells (~ 40%) and CD4+ FoxP3+ T-cells (~ 10%) were consistent across patients and indications. Intriguingly, both stimulatory (CD25, CD28, 4-1BB, ICOS, OX40) and inhibitory (PD-1, CTLA-4, PD-L1, CD39 and TIGIT) checkpoint proteins were predominantly co-expressed by intratumoral CD4+FoxP3+ T-cells. ScRNA-Seq paired with TCR sequencing revealed that T-cells with high clonality and high ICP expressions comprised over 80% of FoxP3+ cells among CD4+ T-cells. Unsupervised clustering of flow cytometry and scRNAseq data identified subsets of CD8+ T-cells and of CD4+ FoxP3- T-cells expressing certain checkpoints, though these expressions were generally lower than in CD4+ FoxP3+ T-cell subsets, both in terms of proportions among total T-cells and ICP expression levels. CONCLUSIONS: Tumor histology alone does not reveal the complete picture of the tumor immune contexture. In clinical trials, assumptions regarding target expression should rely on more sensitive and specific techniques than conventional IHC or transcriptomics. Flow cytometry and scRNAseq accurately characterize ICP expression within immune cell subsets. Much like in hematology, flow cytometry can better describe the immune contexture of solid tumors, offering the opportunity to guide patient treatment according to drug target expression rather than tumor histological type.

Probiotic-guided CAR-T cells for solid tumor targeting.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

From mucosal infection to successful cancer immunotherapy.

The clinical management of advanced malignancies of the upper and lower urinary tract has been revolutionized with the advent of immune checkpoint blockers (ICBs). ICBs reinstate or bolster pre-existing immune responses while creating new T cell specificities. Immunogenic cancers, which tend to benefit more from immunotherapy than cold tumours, harbour tumour-specific neoantigens, often associated with a high tumour mutational burden, as well as CD8+ T cell infiltrates and ectopic lymphoid structures. The identification of beneficial non-self tumour antigens and natural adjuvants is the focus of current investigation. Moreover, growing evidence suggests that urinary or intestinal commensals, BCG and uropathogenic Escherichia coli influence long-term responses in patients with kidney or bladder cancer treated with ICBs. Bacteria infecting urothelium could be a prominent target for T follicular helper cells and B cells, linking innate and cognate CD8+ memory responses. In the urinary tract, commensal flora differ between healthy and tumoural mucosae. Although antibiotics can affect the prognosis of urinary tract malignancies, bacteria can have a major influence on cancer immunosurveillance. Beyond their role as biomarkers, immune responses against uropathogenic commensals could be harnessed for the design of future immunoadjuvants that can be advantageously combined with ICBs.

Neo-Adjuvant immunotherapies: Bladder cancer as a platform for drug development targeting mucosal immunity.

Bacillus Calmette-Guerin (BCG) is a live attenuated Mycobacterium bovis strain, originally developed as a vaccine against tuberculosis. It is also the only bacterial cancer therapy approved by the US Food & Drug Administration for clinical use. BCG is delivered in the bladder, shortly after tumour resection, for patients with high-risk non-muscle invasive bladder cancer (NMIBC). Modulating mucosal immunity by exposing the urothelium to intravesical BCG has been the main therapeutic strategy for high-risk NMIBC over the last three decades. Thus, BCG provides a benchmark for the clinical development of bacteria-or other live attenuated pathogens-as cancer therapy. Currently, a myriad of immuno-oncology compounds is under clinical evaluation in BCG-unresponsive and BCG-naïve patients as an alternative therapy in the context of worldwide BCG shortages. For patients with non-metastatic muscle-invasive bladder cancer (MIBC), studies investigating neoadjuvant immunotherapy with either anti-PD-1/PD-L1 monoclonal antibodies in monotherapy or in combination with anti-CTLA-4 monoclonal antibodies have shown overall efficacy and acceptable safety profiles prior to radical cystectomy. Emerging clinical investigations are testing synergistic approaches by combining intravesical delivery of drugs with systemic immune checkpoint blockades in the neoadjuvant setting for patients with MIBC. Such novel strategy aims to prime a local anti-tumour immunity and reduce distant metastatic relapses by enhancing a systemic adaptive anti-tumour immune response. Here, we present and discuss some of the most promising clinical trials developing such novel therapeutic approaches.

Genomic Instability and Protumoral Inflammation Are Associated with Primary Resistance to Anti-PD-1 + Antiangiogenesis in Malignant Pleural Mesothelioma.

Cancer immunotherapy combinations have recently been shown to improve the overall survival of advanced mesotheliomas, especially for patients responding to those treatments. We aimed to characterize the biological correlates of malignant pleural mesotheliomas' primary resistance to immunotherapy and antiangiogenics by testing the combination of pembrolizumab, an anti-PD-1 antibody, and nintedanib, a pan-antiangiogenic tyrosine kinase inhibitor, in the multicenter PEMBIB trial (NCT02856425). Thirty patients with advanced malignant pleural mesothelioma were treated and explored. Unexpectedly, we found that refractory patients were actively recruiting CD3+CD8+ cytotoxic T cells in their tumors through CXCL9 tumor release upon treatment. However, these patients displayed high levels of somatic copy-number alterations in their tumors that correlated with high blood and tumor levels of IL6 and CXCL8. Those proinflammatory cytokines resulted in higher tumor secretion of VEGF and tumor enrichment in regulatory T cells. Advanced mesothelioma should further benefit from stratified combination therapies adapted to their tumor biology. SIGNIFICANCE: Sequential explorations of fresh tumor biopsies demonstrated that mesothelioma resistance to anti-PD-1 + antiangiogenics is not due to a lack of tumor T-cell infiltration but rather due to adaptive immunosuppressive pathways by tumors, involving molecules (e.g., IL6, CXCL8, VEGF, and CTLA4) that are amenable to targeted therapies. This article is highlighted in the In This Issue feature, p. 799.

Rationale for LDH-targeted cancer immunotherapy.

Immunotherapies have significantly improved the survival of patients in many cancers over the last decade. However, primary and secondary resistances are encountered in most patients. Unravelling resistance mechanisms to cancer immunotherapies is an area of active investigation. Elevated levels of circulating enzyme lactate dehydrogenase (LDH) have been historically considered in oncology as a marker of bad prognosis, usually attributed to elevated tumour burden and cancer metabolism. Recent evidence suggests that elevated LDH levels could be independent from tumour burden and contain a negative predictive value, which could help in guiding treatment strategies in immuno-oncology. In this review, we decipher the rationale supporting the potential of LDH-targeted therapeutic strategies to tackle the direct immunosuppressive effects of LDH on a wide range of immune cells, and enhance the survival of patients treated with cancer immunotherapies.

A machine-learning-based combination of criteria to detect bladder cancer lymph node metastasis on [18F]FDG PET/CT: a pathology-controlled study.

OBJECTIVES: Initial pelvic lymph node (LN) staging is pivotal for treatment planification in patients with muscle-invasive bladder cancer (MIBC), but [18F]FDG PET/CT provides insufficient and variable diagnostic performance. We aimed to develop and validate a machine-learning-based combination of criteria on [18F]FDG PET/CT to accurately identify pelvic LN involvement in bladder cancer patients. METHODS: Consecutive patients with localized MIBC who performed preoperative [18F]FDG PET/CT between 2010 and 2017 were retrospectively assigned to training (n = 129) and validation (n = 44) sets. The reference standard was the pathological status after extended pelvic LN dissection. In the training set, a random forest algorithm identified the combination of criteria that best predicted LN status. The diagnostic performances (AUC) and interrater agreement of this combination of criteria were compared to a consensus of experts. RESULTS: The overall prevalence of pelvic LN involvement was 24% (n = 41/173). In the training set, the top 3 features were derived from pelvic LNs (SUVmax of the most intense LN, and product of diameters of the largest LN) and primary bladder tumor (product of diameters). In the validation set, diagnostic performance did not differ significantly between the combination of criteria (AUC = 0.59 95%CI [0.43-0.73]) and the consensus of experts (AUC = 0.64 95%CI [0.48-0.78], p = 0.54). The interrater agreement was equally good with Κ = 0.66 for both. CONCLUSION: The developed machine-learning-based combination of criteria performs as well as a consensus of experts to detect pelvic LN involvement on [18F]FDG PET/CT in patients with MIBC. KEY POINTS: • The developed machine-learning-based combination of criteria performs as well as experts to detect pelvic LN involvement on [18F]FDG PET/CT in patients with muscle-invasive bladder cancer. • The top 3 features to predict LN involvement were the SUVmax of the most intense LN, the product of diameters of the largest LN, and the product of diameters of the primary bladder tumor.

High serum LDH and liver metastases are the dominant predictors of primary cancer resistance to anti-PD(L)1 immunotherapy.

AIM: Anti-PD-(L)1 immunotherapies improve survival in multiple cancers but remain ineffective for most patients. We applied machine-learning algorithms and multivariate analyses on baseline medical data to estimate their relative impact on overall survival (OS) upon anti-PD-(L)1 monotherapies. METHOD: This prognostic/predictive study retrospectively analysed 33 baseline routine medical variables derived from computed tomography (CT) images, clinical and biological meta-data. 695 patients with a diagnosis of advanced cancer were treated in prospective clinical trials in a single tertiary cancer centre in 3 cohorts including systemic anti-PD-(L)1 (251, 235 patients) versus other systemic therapies (209 patients). A random forest model combined variables to identify the combination (signature) which best estimated OS in patients treated with immunotherapy. The performance for estimating OS [95%CI] was measured using Kaplan-Meier Analysis and Log-Rank test. RESULTS: Elevated serum lactate dehydrogenase (LDHhi) and presence of liver metastases (LM+) were dominant and independent predictors of short OS in independent cohorts of melanoma and non-melanoma solid tumours. Overall, LDHhiLM+ patients treated with anti-PD-(L)1 monotherapy had a poorer outcome (median OS: 3.1[2.4-7.8] months]) compared to LDHlowLM-patients (median OS: 15.3[8.9-NA] months; P < 0.0001). The OS of LDHlowLM-patients treated with immunotherapy was 28.8[17.9-NA] months (vs 13.1[10.8-18.5], P = 0.02) in the overall population and 30.3[19.93-NA] months (vs 14.1[8.69-NA], P = 0.0013) in patients with melanoma. CONCLUSION: LDHhiLM+ status identifies patients who shall not benefit from anti-PD-(L)1 monotherapy. It could be used in clinical trials to stratify patients and eventually address this specific medical need.

Escherichia coli-Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer.

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale. SIGNIFICANCE: In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli-specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221.

BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer.

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guérin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with HLA-I-deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with HLA-I-proficient cancer cells after BCG therapy presented with CD8+ T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint-inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer.

Plasmacytoid urothelial carcinoma (UC) are luminal tumors with similar CD8+ Tcell density and PD-L1 protein expression on immune cells as compared to conventional UC.

BACKGROUND: Plasmacytoid urothelial carcinoma (UC) is a rare pathological variant of UC with low chemotherapeutic sensitivity and dismal outcomes. The molecular and immune profiles of such tumors remain poorly investigated. METHODS: Herein, we investigated the phenotypical features of a cohort of plasmacytoid UC (n=32) by comparison to a control group of conventional high-grade UC with matched clinicopathological characteristics (n=30). Histopathological analysis included the following antibodies: p63, GATA3, CK5/6, CK20 and HER2. In addition, the density of intra-tumor CD8+ lymphocytes, and PD-L1 expression in tumor (TC) and immune cells (IC) were evaluated. RESULTS: Plasmacytoid UC expressed GATA3 (97% vs 86% P=0.18), CK20 (59% vs 36% P=0.08) markers and showed a significantly higher rate of HER2 overexpression (2+ and 3+ score: 25% vs 0%, P<0.01) compared to controls. A significantly lower expression of CK5/6 (22% vs 56%, P<0.05) and p63 (41% vs 80%, P<0.05) was observed in plasmacytoid UC compared to controls. The density of tumor-infiltrating CD8+ cells was similar between plasmacytoid and conventional UC (P=0.9). PD-L1 expression on IC was similar compared to conventional UC (P=0.3). CONCLUSIONS: Together, our study demonstrated that plasmacytoid UC belong to the luminal subtype and display a rather inflamed microenvironment similar to conventional UC. These data support the inclusion of plasmacytoid variant of UC in clinical trials evaluating immune checkpoint inhibitors monotherapy or combination immunotherapeutic strategies.

Targeting the DNA damage response in immuno-oncology: developments and opportunities.

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcome with regard to multiple tumour types. However, most patients still do not benefit from such therapies, notably because of the absence of pre-existing T cell infiltration. DNA damage response (DDR) deficiency has recently emerged as an important determinant of tumour immunogenicity. A growing body of evidence now supports the concept that DDR-targeted therapies can increase the antitumour immune response by (1) promoting antigenicity through increased mutability and genomic instability, (2) enhancing adjuvanticity through the activation of cytosolic immunity and immunogenic cell death and (3) favouring reactogenicity through the modulation of factors that control the tumour-immune cell synapse. In this Review, we discuss the interplay between the DDR and anticancer immunity and highlight how this dynamic interaction contributes to shaping tumour immunogenicity. We also review the most innovative preclinical approaches that could be used to investigate such effects, including recently developed ex vivo systems. Finally, we highlight the therapeutic opportunities presented by the exploitation of the DDR-anticancer immunity interplay, with a focus on those in early-phase clinical development.

Relationship of preoperative androgen levels and metabolic syndrome with quality of life and erectile function in patients who are to undergo radical prostatectomy.

This study aims to investigate whether clinical and biological preoperative characteristics of patients who were to undergo radical prostatectomy were associated with impairment in patient-reported quality of life (QoL) and erectile dysfunction immediately before intervention. We evaluated patient-reported outcomes among 1019 patients (out of 1343) of the AndroCan study, willing to score the Aging Male Symptom (AMS) and the International Index of Erectile Function 5-item (IIEF-5) auto-questionnaires. Univariate linear regression and robust multiple regression were used to ascertain the relationship between demographic, clinical, and hormonal parameters and global AMS or IIEF-5 scores. As a result, most patients (85.1') of the Androcan cohort agreed to complete questionnaires. Significantly higher IIEF-5 global scores were found in non-Caucasian and obese patients, with larger waist circumference, metabolic syndrome, diabetes mellitus, cardiovascular disease, hypertension, high blood sugar, concomitant medications, and hypogonadism, while the AMS global score was significantly higher in patients with larger waist circumference, metabolic syndrome, high blood pressure, raised glycemia, and concomitant medication. The IIEF-5 global score was correlated to age, dehydroepiandrosterone (DHEA), fat mass percentage, and androstenediol (D5). The AMS global score was significantly correlated to DHEA, D5, and DHEA sulfate. Finally, the multivariate models showed that QoL and erectile function were significantly affected, before surgery, by symptoms and signs that are usually considered as pertaining to the metabolic syndrome, while sexual hormones are essentially correlated to erectile dysfunction.