The estrogen signaling pathway reprograms prostate cancer cell metabolism and supports proliferation and disease progression.

Just as the androgen receptor (AR), the estrogen receptor α (ERα) is expressed in the prostate and is thought to influence prostate cancer (PCa) biology. Yet, the incomplete understanding of ERα functions in PCa hinders our ability to fully comprehend its clinical relevance and restricts the repurposing of estrogen-targeted therapies for the treatment of this disease. Using two human PCa tissue microarray cohorts, we first demonstrated that nuclear ERα expression was heterogeneous among patients, being only detected in half of tumors. Positive nuclear ERα levels were correlated with disease recurrence, progression to metastatic PCa, and patient survival. Using in vitro and in vivo models of the normal prostate and PCa, bulk and single-cell RNA-Seq analyses revealed that estrogens partially mimic the androgen transcriptional response and induce specific biological pathways linked to proliferation and metabolism. Bioenergetic flux assays and metabolomics confirmed the regulation of cancer metabolism by estrogens, supporting proliferation. Using cancer cell lines and patient-derived organoids, selective estrogen receptor modulators, a pure anti-estrogen, and genetic approaches impaired cancer cell proliferation and growth in an ERα-dependent manner. Overall, our study revealed that, when expressed, ERα functionally reprograms PCa metabolism, is associated with disease progression, and could be targeted for therapeutic purposes.

T follicular helper cells expansion in transplant recipients correlates with graft infiltration and adverse outcomes.

Introduction: The process of immunization following vaccination in humans bears similarities to that of immunization with allografts. Whereas vaccination aims to elicit a rapid response, in the transplant recipient, immunosuppressants slow the immunization to alloantigens. The induction of CD4+CXCR5+ T follicular helper (Tfh) cells has been shown to correlate with the success of vaccine immunization. Method: We studied a cohort of 65 transplant recipients who underwent histological evaluation concurrent with PBMC isolation and follow-up sampling to investigate the phenotypic profiles in the blood and allotissue and analyze their association with clinical events. Results: The proportion of circulating Tfh cells was heterogeneous over time. Patients in whom this compartment increased had lower CCR7-PD1+CD4+CXCR5+ T cells during follow-up. These patients exhibited more alloreactive CD4+ T cells using HLA-DR-specific tetramers and a greater proportion of detectable circulating plasmablasts than the controls. Examination of baseline biopsies revealed that expansion of the circulating Tfh compartment did not follow prior intragraft leukocyte infiltration. However, multicolor immunofluorescence microscopy of the grafts showed a greater proportion of CXCR5+ T cells than in the controls. CD4+CXCR5+ cells were predominantly PD1+ and were in close contact with B cells in situ. Despite clinical stability at baseline, circulating Tfh expansion was associated with a higher risk of a composite of anti-HLA donor-specific antibodies, rejection, lower graft function, or graft loss. Conclusion: In otherwise stable patients post-transplant, circulating Tfh expansion can identify ongoing alloreactivity, detectable before allograft injury. Tfh expansion is relevant clinically because it predicts poor graft prognosis. These findings have implications for immune surveillance.

Isocitrate dehydrogenase 1 sustains a hybrid cytoplasmic-mitochondrial tricarboxylic acid cycle that can be targeted for therapeutic purposes in prostate cancer.

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

Expression of Class II Human Leukocyte Antigens on Human Endothelial Cells Shows High Interindividual and Intersubclass Heterogeneity.

SIGNIFICANCE STATEMENT: Donor-specific antibodies against class II HLA are a major cause of chronic kidney graft rejection. Nonetheless, some patients presenting with these antibodies remain in stable histological and clinical condition. This study describes the use of endothelial colony-forming cell lines to test the hypothesis of the heterogeneous expression of HLA molecules on endothelial cells in humans. Flow cytometry and immunofluorescence staining revealed substantial interindividual and interlocus variability, with HLA-DQ the most variable. Our data suggest that the expression of HLA class II is predicted by locus. The measurement of endothelial expression of HLA class II in the graft could present a novel paradigm in the evaluation of the alloimmune risk in transplantation and certain diseases. BACKGROUND: HLA antigens are important targets of alloantibodies and allospecific T cells involved in graft rejection. Compared with research into understanding alloantibody development, little is known about the variability in expression of their ligands on endothelial cells. We hypothesized individual variability in the expression of HLA molecules. METHODS: We generated endothelial colony forming cell lines from human peripheral blood mononuclear cells ( n =39). Flow cytometry and immunofluorescence staining were used to analyze the cells, and we assessed the relationship between HLA-DQ expression and genotype. Two cohorts of kidney transplant recipients were analyzed to correlate HLA-DQ mismatches with the extent of intragraft microvascular injury. RESULTS: Large variability was observed in the expression of HLA class II antigens, not only between individuals but also between subclasses. In particular, HLA-DQ antigens had a low and heterogeneous expression, ranging from 0% to 85% positive cells. On a within-patient basis, this expression was consistent between endothelial cell colonies and antigen-presenting cells. HLA-DQ5 and -DQ6 were associated with higher levels of expression, whereas HLA-DQ7, -DQ8, and -DQ9 with lower. HLA-DQ5 mismatches among kidney transplant recipients were associated with significant increase in graft microvascular. CONCLUSION: These data challenge the current paradigm that HLA antigens, in particular HLA class II, are a single genetic and post-translational entity. Understanding and assessing the variability in the expression of HLA antigens could have clinical monitoring and treatment applications in transplantation, autoimmune diseases, and oncology.

The Progression of Interstitial Fibrosis and Tubular Atrophy at 6 Months Is an Independent Predictor of Poor Graft Outcomes in Kidney Transplant Recipients.

Interstitial fibrosis and tubular atrophy (IFTA) found on 1-y surveillance biopsies has been associated with poor graft outcomes. However, its progression over time and relationship to outcomes are less well defined. Methods: We studied implantation and 6-mo surveillance biopsies and examined the association between the progression of IFTA (ΔIFTA) and a composite of censored graft loss or doubling of serum creatinine in 248 adult kidney recipients. Results: The percentage of patients with ΔIFTA of 1 or ≥2 was 35% and 22%, respectively. Positive ΔIFTA was a risk factor for the composite endpoint (hazard ratio, 1.36; 95% confidence interval, 1.03-1.79). This estimate was robust to adjustment for recipient and donor baseline characteristics, baseline IFTA, tacrolimus levels, and rejection status. ΔIFTA was associated with decreased estimated glomerular filtration rate at 3 and 5 y. IFTA+i was a predictor in the cohort; however, IFTA progression was not limited to those with a mononuclear cell interstitial inflammation (Banff "i") score above zero. Notably, donor age was a predictor of IFTA at 6 mo, but not of ΔIFTA, whereas rejection, donor diabetes, and recipient smoking status were. Conclusions: Progression of IFTA at 6 mo can predict outcomes. ΔIFTA was not related to donor age but may be linked to other risk factors influencing decision-making for donor versus recipient selection.

Association of HLA Typing and Alloimmunity With Posttransplantation Membranous Nephropathy: A Multicenter Case Series.

RATIONALE & OBJECTIVES: Posttransplantation membranous nephropathy (MN) represents a rare complication of kidney transplantation that can be classified as recurrent or de novo. The clinical, pathologic, and immunogenetic characteristics of posttransplantation MN and the differences between de novo and recurrent MN are not well understood. STUDY DESIGN: Multicenter case series. SETTING & PARTICIPANTS: We included 77 patients from 5 North American and European medical centers with post-kidney transplantation MN (27 de novo and 50 recurrent). Patients with MN in the native kidney who received kidney allografts but did not develop recurrent MN were used as nonrecurrent controls (n = 43). To improve understanding of posttransplantation MN, we compared de novo MN with recurrent MN and then contrasted recurrent MN with nonrecurrent controls. FINDINGS: Compared with recurrent MN, de novo MN was less likely to be classified as primary MN (OR, 0.04; P < 0.001) and had more concurrent antibody-mediated rejection (OR, 12.0; P < 0.001) and inferior allograft survival (HR for allograft failure, 3.2; P = 0.007). HLA-DQ2 and HLA-DR17 antigens were more common in recipients with recurrent MN compared with those with de novo MN; however, the frequency of these recipient antigens in recurrent MN was similar to that in nonrecurrent MN controls. Among the 93 kidney transplant recipients with native kidney failure attributed to MN, older recipient age (HR per each year older, 1.03; P = 0.02), recipient HLA-A3 antigen (HR, 2.5; P = 0.003), steroid-free immunosuppressive regimens (HR, 2.84; P < 0.001), and living related allograft (HR, 1.94; P = 0.03) were predictors of MN recurrence. LIMITATIONS: Retrospective case series, limited sample size due to rarity of the disease, nonstandardized nature of data collection and biopsies. CONCLUSIONS: De novo and recurrent MN likely represent separate diseases. De novo MN is associated with humoral alloimmunity and guarded outcome. Potential predisposing factors for recurrent MN include recipients who are older, recipient HLA-A3 antigen, steroid-free immunosuppressive regimen, and living related donor kidney.

Impact of the Current Versus the Previous Diagnostic Threshold on the Outcome of Patients With Borderline Changes Suspicious for T Cell-mediated Rejection Diagnosed on Indication Biopsies.

BACKGROUND: Since the borderline changes suspicious for acute T cell-mediated rejection (BL) category was broadened, there has been a debate regarding the right threshold for tubulitis and interstitial inflammation scores. METHODS: We studied a first cohort of 111 patients with BL found on an indication biopsy between 2006 and 2016 and compared those with scores of t1i0 (BLt1i0) to those with higher scores (BL≥t1i1). A second cohort of 56 patients with BL was used for external validation. We used a composite endpoint of death-censored graft failure or doubling of the serum creatinine level postbiopsy. RESULTS: In the first cohort, 68% (75/111) of the BL cases fell in the BLt1i0 group. At 5 years, the occurrence of the composite endpoint was 5% and 14% for BLt1i0 and BL≥t1i1, respectively. In contrast, the endpoint occurred in 5% of nonrejectors and 21% of patients with T cell-mediated rejection. In the validation cohort, 8% versus 36% of BLt1i0 and BL≥t1i1 reached the endpoint, respectively. Multivariable Cox modeling revealed that BLt1i0 patients had a prognosis similar to that of nonrejectors (adjusted hazard ratio, 0.6; 95% confidence interval, 0.1-2.2; P = 0.40) but better than that of patients with BL≥t1i1 (hazard ratio, 3.8; 95% confidence interval, 1.3-11.5; P = 0.02). Sensitivity analyses restricted to death-censored graft loss or using time posttransplant as the time of reference provided similar results. CONCLUSIONS: In summary, patients with BLt1i0 have a different prognosis to that of BL≥t1i1 patients, which brings into question the current diagnostic thresholds.

Allogeneic dendritic cells stimulated with antibodies against HLA class II polarize naive T cells in a follicular helper phenotype.

Follicular helper T cells (Tfh) are crucial for the production of high-affinity antibodies, such as alloantibodies, by providing the signals for B-cell proliferation and differentiation. Here, we demonstrate that human allogeneic dendritic cells (DC) stimulated with antibodies against HLA class II antigens preferentially differentiate human naive CD4+ T cells into Tfh cells. Following coculture with DCs treated with these antibodies, CD4+ T cells expressed CXCR5, ICOS, IL-21, Bcl-6 and phosphorylated STAT3. Blockade of IL-21 abrogated Bcl-6, while addition of the IL-12p40 subunit to the coculture increased CXCR5, Bcl-6, phosphorylated STAT3 and ICOS, indicating that they were both involved in Tfh polarization. We further phenotyped the peripheral T cells in a cohort of 55 kidney transplant recipients. Patients with anti-HLA-II donor-specific antibodies (DSA) presented higher blood counts of circulating Tfh cells than those with anti-HLA-I DSAs. Moreover, there was a predominance of lymphoid aggregates containing Tfh cells in biopsies from patients with antibody-mediated rejection and anti-HLA-II DSAs. Collectively, these data suggest that alloantibodies against HLA class II specifically promote the differentiation of naive T cells to Tfh cells following contact with DCs, a process that might appear in situ in human allografts and constitutes a therapeutic target.

IL-6 production by monocytes is associated with graft function decline in patients with borderline changes suspicious for acute T-cell-mediated rejection: a pilot study.

Although borderline changes (BL) suspicious for acute T-cell-mediated rejection represent a diagnostic category, its clinical relevance is questioned leading to heterogeneous therapeutic management. We hypothesized that measuring IL-6 secretion by peripheral blood mononuclear cells identifies patients with ongoing graft damage. We examined the association between secreted IL-6 and the change in estimated glomerular filtration rate at 6 months after the biopsy (ΔeGFR). We then conducted phenotypic and functional studies on patient and mouse innate immune cells in the blood and the kidney. In a training set, ΔeGFR was strongly associated with IL-6 levels, showing a clinically meaningful decline of 4.6 ± 1.5 ml/min per increase in log10 IL-6 (P = 0.001). These results were consistent after adjustment and were reproduced in a validation cohort. Phenotyping of peripheral blood cells revealed that the main source of IL-6 was CD14+ CD16- CCR2+ HLA-DR+ CD86+ CD11c+ inflammatory monocytes. There was a significant correlation between IL-6 secretion and interstitial dendritic cell density in the biopsy. Finally, characterization of mouse kidney dendritic cells revealed that they share features with macrophages and function as effector cells secreting IL-6. In conclusion, measuring IL-6 secreted by peripheral blood cells can be useful in the management of patients with BL in the absence of a concurrent inflammatory condition.

Higher calcineurin inhibitor levels predict better kidney graft survival in patients with de novo donor-specific anti-HLA antibodies: a cohort study.

The development of de novo anti-HLA donor-specific antibodies (dnDSA) is associated with poorer outcomes in kidney transplant recipients. Despite this, antibody screening post-transplant is not widespread, largely because the optimal management of patients with dnDSA remains undetermined. We hypothesized that in this population, calcineurin inhibitor blood levels would be an independent predictor of graft loss. We analyzed a cohort of unsensitized patients for whom anti-HLA antibody screening was performed prospectively post-transplant. During the screening period between January 2005 and April 2016, 42 patients developed dnDSA. There was no difference in the clinical characteristics or the histological scores of patients biopsied for clinical indication versus those biopsied solely due to detection of dnDSA. Cox modeling revealed a strong relationship between mean tacrolimus levels following dnDSA detection and graft loss, with a hazard ratio of 0.49 (95% CI, 0.33-0.75), which persisted following adjustment for established independent predictors (HR, 0.52, 95% CI, 0.30-0.89). Kaplan-Meier analysis by tertiles of tacrolimus levels and receiver operating curve analysis concurred to show that a threshold of 5.3 ng/ml could be predictive of graft loss. These data suggest that anti-HLA antibody monitoring post-transplant could guide maintenance immunosuppression and improve graft outcomes.

Improved cancer specific-survival in patients with carcinoma invading bladder muscle expressing cyclo-oxygenase-2.

UNLABELLED: Study Type - Prognosis (case series). LEVEL OF EVIDENCE: 4 OBJECTIVE: To determine whether the expression of cyclo-oxygenase (COX)-2 has an influence on survival and on the response to chemotherapy in invasive bladder cancer. PATIENTS AND METHODS: A population of 266 patients from a tertiary university centre with carcinoma invading bladder muscle without evidence of metastasis at time of cystectomy was analyzed retrospectively. COX-2 expression was evaluated immunohistochemically with a monoclonal anti-COX-2 antibody. All pertinent clinical and pathological parameters were reviewed and correlated with risk factors influencing outcome, including disease-specific and overall survival, as well as COX-2 expression. Immunoreactivity was categorized as positive if COX-2 staining was present in >5% tumour cells. RESULTS: The expression of COX-2 was not influenced by tumour stage, grade or nodal status, nor any other parameters. The risk factors that influenced disease-specific survival in carcinoma invading bladder muscle on multivariate analysis were lymph node status (hazards ratio, HR = 2.46 for N1, P = 0.001, HR = 2.90 for N2, P < 0.001, HR = 5.19 for N3, P = 0.012), use of neoadjuvant chemotherapy (HR = 3.54; P= 0.004) or adjuvant chemotherapy (HR = 0.57, P = 0.014) and COX-2 expression (HR = 0.64 if >5% cells had positive expression; P = 0.025). Kaplan-Meier analysis showed an increased disease-specific survival (P = 0.0063), as well as longer recurrence-free survival (P = 0.003), in patients with muscle-invasive bladder tumours expressing COX-2 in >5% of the cells. A tendency was also observed in a subgroup with positive nodes treated with adjuvant chemotherapy (P = 0.093). CONCLUSIONS: The overexpression of COX-2 is associated with a better recurrence-free and disease-specific survival in a large cohort of 266 patients with carcinoma invading bladder muscle treated by cystectomy. A trend for increased disease-specific survival was also observed for patients with COX-2 overexpression and positive nodes who received adjuvant chemotherapy. Potential of COX-2 as a prognostic marker in bladder cancer should be considered.

Screening for polyomavirus associated nephropathy in renal transplantation with blood viral load measurement.

BACKGROUND: Polyomavirus associated nephropathy (PVAN) is an important cause of graft failure in the renal transplant population. It has been shown that viremia precedes PVAN, suggesting that measurement of blood viral load could be used for PVAN screening. OBJECTIVES: To verify the utility of BK virus (BKV) blood viral load measurement for PVAN screening in the renal transplant population, establish a threshold value, and determine the sensitivity and specificity of the test. STUDY DESIGN: We developed a real-time PCR assay for BKV blood viral load measurement and included this assay in the PVAN screening protocol of the renal transplant recipients of our institution. We report results for 60 patients who had a blood viral load measurement concomitantly with an allograft biopsy with immunohistochemistry for polyomavirus. RESULTS: 14 patients were found to have a PVAN on allograft biopsy together with a viral load above 3.0x10(3)copies/ml. None of the patients with a viral load under 3.0x10(3)copies/ml had a PVAN on allograft biopsy. The area under the receiver operating characteristic (ROC) curve was 0.95 (95% CI: 0.91-1.00) and using a threshold value of 3.0x10(3)copies/ml yielded a sensitivity of 100% (95% CI: 76.8-100%) and a specificity of 89.6% (95% CI: 77.3-96.5%) for PVAN screening. CONCLUSIONS: BKV blood viral load measurement is sensitive and specific for PVAN screening when a threshold value is precisely determined.

Accelerated induction of bladder cancer in patched heterozygous mutant mice.

The PATCHED (PTC) gene is recognized as a tumor suppressor in basal cell carcinoma. Mapping of a minimal region of deletion at 9q22.3 and observation of a decreased PTC expression in superficial papillary bladder tumors led us to hypothesize that it could also be involved in this cancer. To further investigate this hypothesis, we submitted Ptc(+/-) heterozygous mutant mice and their wild-type littermates to chemical carcinogenesis by adding N-butyl-N-(4-hydroxybutyl) nitrosamine to their drinking water. Preneoplastic and neoplastic changes were observed significantly earlier in the Ptc(+/-) than in the wild-type mice. Our data support the hypothesis of Ptc acting as a tumor suppressor gene in bladder cancer.

Expression of p21 cell cycle protein is an independent predictor of response to salvage radiotherapy after radical prostatectomy.

BACKGROUND: To assess whether the expression of p21, p27, and p53 could predict biochemical failure in prostate cancer patients treated with neoadjuvant androgen deprivation prior to salvage radiotherapy for a rising post-radical prostatectomy (RP) prostate-specific antigen (PSA). METHODS: The expression of p21, p27, and p53 was determined by immunohistochemistry in a cohort of 74 formalin-fixed paraffin-embedded prostate cancer samples obtained from RP. Expression of these markers was then correlated with clinicopathological parameters and biochemical failure-free survival after salvage radiotherapy. RESULTS: Expression of p21, p27, and p53 was observed in 20%, 69%, and 74% of prostate cancer specimens, respectively. Overexpression of p21 correlated with a higher Gleason score (>7) (P = 0.024). Of the three markers, only p21 expression was correlated with PSA failure after radiotherapy (P = 0.034). In multivariate analysis, both positive p21 (P = 0.004) and pre-radiation serum PSA > 1 ng/ml (P < 0.0001) were independent predictors of biochemical failure after salvage radiotherapy. Patients with p21- tumors and a serum PSA level < or = 1 ng/ml before salvage radiotherapy had a biochemical failure-free survival at 5 years of 83%, compared to 16% at 5 years for those patients with either p21+ tumor or a PSA > 1 ng/ml. Patients with both p21+ and a PSA level > 1 ng/ml had a much lower biochemical failure-free survival rate of 25% at only 18 months (P < 0.0001). CONCLUSIONS: The expression of p21 in prostatectomy specimens could help predict the likelihood of response to salvage radiotherapy, particularly in patients treated before PSA reaches 1 ng/ml.

Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance.

The androgen-signaling pathway is critical to the development and progression of prostate cancer and androgen ablation is a mainstay of therapy for this disease. We performed a genome-wide expression analysis of human prostate cancer during androgen ablation therapy to identify genes regulated by androgen and genes differentially expressed after the development of resistance. Six hundred and fifty-four of 63,175 probe sets detected significant expression changes after 3 months of treatment with goserelin and flutamide. This included 149 genes that were also differentially expressed 36 hours after androgen withdrawal in LNCaP cells. These genes reflect the physiological changes that occur in treated tumors and include potential direct targets of the androgen receptor. Expression profiles of androgen ablation-resistant tumors demonstrated that many of the gene expression changes detected during therapy were no longer present suggesting a reactivation of the androgen response pathway in the absence of exogenous hormone. Therapy resistance was associated with differential expression of a unique set of genes that reflect potential mechanisms of reactivation. Specifically an up-regulation of the androgen receptor and key enzymes for steroid biosynthesis suggest that resistant tumors have increased sensitivity to and endogenous synthesis of androgenic hormones. The specific pathways of reactivation provide opportunities for classification of resistant tumors and targeted therapies.

Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease.

The identification of genes that contribute to the biological basis for clinical heterogeneity and progression of prostate cancer is critical to accurate classification and appropriate therapy. We performed a comprehensive gene expression analysis of prostate cancer using oligonucleotide arrays with 63,175 probe sets to identify genes and expressed sequences with strong and uniform differential expression between nonrecurrent primary prostate cancers and metastatic prostate cancers. The mean expression value for >3,000 tumor-intrinsic genes differed by at least 3-fold between the two groups. This includes many novel ESTs not previously implicated in prostate cancer progression. Many differentially expressed genes participate in biological processes that may contribute to the clinical phenotype. One example was a strong correlation between high proliferation rates in metastatic cancers and overexpression of genes that participate in cell cycle regulation, DNA replication, and DNA repair. Other functional categories of differentially expressed genes included transcriptional regulation, signaling, signal transduction, cell structure, and motility. These differentially expressed genes reflect critical cellular activities that contribute to clinical heterogeneity and provide diagnostic and therapeutic targets.