Artificial intelligence to improve cytology performance in urothelial carcinoma diagnosis: results from validation phase of the French, multicenter, prospective VISIOCYT1 trial.

PURPOSE: Cytology and cystoscopy, the current gold standard for diagnosing urothelial carcinomas, have limits: cytology has high interobserver variability with moderate or not optimal sensitivity (particularly for low-grade tumors); while cystoscopy is expensive, invasive, and operator dependent. The VISIOCYT1 study assessed the benefit of VisioCyt® for diagnosing urothelial carcinoma. METHODS: VISIOCYT1 was a French prospective clinical trial conducted in 14 centers. The trial enrolled adults undergoing endoscopy for suspected bladder cancer or to explore the lower urinary tract. Participants were allocated either Group 1: with bladder cancer, i.e., with positive cystoscopy or with negative cystoscopy but positive cytology, or Group 2: without bladder cancer. Before cystoscopy and histopathology, slides were prepared for cytology and the VisioCyt® test from urine samples. The diagnostic performance of VisioCyt® was assessed using sensitivity (primary objective, 70% lower-bound threshold) and specificity (75% lower-bound threshold). Sensitivity was also assessed by tumor grade and T-staging. VisioCyt® and cytology performance were evaluated relative to the histopathological assessments. RESULTS: Between October 2017 and December 2019, 391 participants (170 in Group 1 and 149 in Group 2) were enrolled. VisioCyt®'s sensitivity was 80.9% (95% CI 73.9-86.4%) and specificity was 61.8% (95% CI 53.4-69.5%). In high-grade tumors, the sensitivity was 93.7% (95% CI 86.0-97.3%) and in low-grade tumors 66.7% (95% CI 55.2-76.5%). Sensitivity by T-staging, compared to the overall sensitivity, was higher in high-grade tumors and lower in low-grade tumors. CONCLUSION: VisioCyt® is a promising diagnostic tool for urothelial cancers with improved sensitivities for high-grade tumors and notably for low-grade tumors.

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.

Artificial intelligence to improve cytology performances in bladder carcinoma detection: results of the VisioCyt test.

OBJECTIVE: To explore the utility of artificial intelligence (AI) using the VisioCyt® test (VitaDX International, Rennes, France) to improve diagnosis of bladder carcinoma using voided urine cytology. PATIENTS AND METHODS: A national prospective multicentre trial (14 centres) was conducted on 1360 patients, divided in two groups. The first group included bladder carcinoma diagnosis with different histological grades and stages, and the second group included control patients based on negative cystoscopy and cytology results. The first step of this VISIOCYT1 trial focussed on algorithm development and the second step on validating this algorithm. A total of 598 patients were included in this first step, 449 patients with bladder tumours (219 high-grade and 230 low-grade) and 149 as negative controls. The VisioCyt test was compared to voided urine cytology performed by experienced uro-pathologists from each centre. RESULTS: Overall sensitivity was highly improved by the VisioCyt test compared to cytology (84.9% vs 43%). For high-grade tumours the VisioCyt test sensitivity was 92.6% vs 61.1% for the uro-pathologists. Regarding low-grade tumours, VisioCyt test sensitivity was 77% vs 26.3% for the uro-pathologists. CONCLUSION: In comparison to routine cytology, the results of the first phase of the VISIOCYT1 trial show very clear progress in terms of sensitivity, which is particularly visible and interesting for low-grade tumours. If the validation cohort confirms these results, it could lead to the VisioCyt test being considered as a very useful aid for pathologists. Moreover, as this test is in fact software based on AI, it should become more and more efficient as more data are collected.

PD-L1 testing in urothelial bladder cancer: essentials of clinical practice.

PURPOSE: While immunotherapy has become an increasingly attractive strategy in patients with urothelial bladder cancer, the need for a biomarker to identify patients whose cancer is the most likely to respond has never been more crucial. This review systematically evaluates evidence regarding PD-L1 as a predictive biomarker of response to anti-PD(L)1 monoclonal antibodies in patients with urothelial bladder carcinoma, and discusses its current limits in routine clinical practice. METHODS: We performed a critical review of PubMed/Medline according to the Preferred Reporting Items for Systematic Review and Meta-analyses (PRISMA) statement. Prospective clinical trials evaluating anti-PD(L)1 monoclonal antibodies in urothelial bladder carcinoma together with retrospective studies evaluating PD-L1 expression in patients with bladder cancer were included. RESULTS: Evidence data related to PD-L1 as a predictive biomarker of response to immune checkpoint blockade monotherapy across clinical trials are detailed in this review. The different companion diagnostic assays, and the methods for PD-L1 scoring in urothelial bladder carcinoma are reported. Additionally, the issues related to the implementation of PD-L1 testing in clinical practice are discussed. CONCLUSIONS: PD-(L)1 monoclonal antibodies atezolizumab and pembrolizumab are restricted to patients with PD-L1 positive status in the first-line setting in patients with advanced or metastatic urothelial bladder carcinoma who are ineligible to cisplatin-based chemotherapy. Importantly, the use of anti-PD(L)1 mAb in the other clinical settings is not based on PD-L1 status, but rather on patients' clinical characteristics. Further identification of biomarkers with high negative predictive value will also be of utmost importance to identify patients who may not respond to such immunotherapies.

Sex steroids in serum and prostatic tissue of human cancerous prostate (STERKPROSER trial).

BACKGROUND: Currently, there is no consensus regarding the expected concentration levels of intra-prostatic sex steroids in patients with Prostate Cancer (PCa). Our objective was to assess the concentration levels of sex steroids in prostatic tissue and serum, in two cohorts of patients with localized PCa or benign prostatic hyperplasia (BPH). METHODS: Between September 2014 and January 2017, men selected for radical cystectomy (for bladder cancer) or open prostatectomy (for BPH), and men selected for radical prostatectomy for localized PCa were included. Blood samples were collected at baseline before surgery, and steroid concentrations were assessed following the recommendations of the Endocrine Society. Intra-prostatic samples were collected from fresh surgical samples, and assessed by gas chromatography and mass spectrometry (GC/MS). Permanova analysis was performed. Analyses were adjusted for age, prostate weight, and prostate-specific antigen (PSA) level. RESULTS: A total of 73 patients (41 patients with PCa and 32 patients with BPH) were included in this study. Patients with PCa were younger, and had smaller prostate volumes with higher levels of PSA. The levels of Total Testosterone (TT), Di-Hydro-Testosterone (DHT), and Estradiol (E2) in the serum were not significantly different between PCa and BPH. In PCa tissue, TT concentrations were significantly lower (0.11 ng/g vs 0.47 ng/g, P = 0.0002), however its derivative E2 had significantly higher concentrations (31.0 ng/g vs 22.3 ng/g, P = 0.01). DHT tissue concentrations were not significantly different between the two groups (5.55 ng/g vs 5.42 ng/g, P = 0.70). Intra-prostatic TT concentrations were significantly lower in the peripheral zone than in the central zone for the CaP group (0.07 ng/g vs 0.15 ng/g, P = 0.004). CONCLUSIONS: Patients with PCa had lower intra-prostatic TT and higher E2 concentrations levels compared to the patients with BPH. PCa seem to consume more TT and produce more E2, especially in the peripheral zone.

Integrated analysis of 18F-FDG PET/CT improves preoperative lymph node staging for patients with invasive bladder cancer.

OBJECTIVES: Preoperative 18F-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) is controversial to assess lymph node (LN) staging in patients with invasive bladder cancer. We proposed to use the maximum standardized uptake value (SUVmax) associated with axial-based LN size to improve the detection of regional LN metastasis. METHODS: From May 2015 to May 2017, we prospectively included patients with urothelial bladder cancer who underwent radical cystectomy with extended pelvic LN dissection. All patients underwent preoperative 18F-FDG PET/CT staging before surgery. The gold standard comparator was the pathological examination of resected LNs. The data were reported on a regional per area- and patient-based model according to SUVmax values and axial-based LN size criteria. RESULTS: In total, 1012 LNs were identified in 61 patients with clinically localized invasive bladder cancer who underwent radical cystectomy and extended pelvic LN dissection. Loco-regional involvement of 24 LN areas was confirmed in 17 patients. In per area analysis, diagnostic accuracy of PET/CT and CT alone were respectively 84% and 78% (p = 0.039). On patient-based analysis, combined PET/CT correctly classified pelvic LN status in 5/61 (+ 8%) additional patients using optimal thresholds compared to CT alone, with accuracies of 82% and 74%, respectively (p = 0.13). CONCLUSION: Combining SUVmax and axial-based LN size criteria using 18F-FDG PET/CT improved the diagnostic accuracy for preoperative LN staging in patients with invasive bladder cancer, in per area analysis. KEY POINTS: • Combining metabolical and morphological features using18F-FDG PET/CT improves the detection of malignant lymph node in patients with bladder cancer. • 18 F-FDG PET/CT may help for initial staging of patients with muscle invasive bladder cancer.

Sexual steroids in serum and prostatic tissue of human non-cancerous prostate (STERPROSER trial).

BACKGROUND: The specific involvement of the sex steroids in the growth of the prostatic tissue remains unclear. Sex steroid concentrations in plasma and in fresh surgical samples of benign central prostate were correlated to prostate volume. METHODS: Monocentric prospective study performed between September 2014 and January 2017. Age, obesity parameters, and both serum and intraprostatic concentrations of sex steroids were collected complying with the latest Endocrine Society guidelines and the steroids assessed by GC/MS. Statistical calculations were adjusted for age and body mass index (BMI). RESULTS: Thirty-two patients, equally divided between normal- and high-volume prostate groups, were included in the analysis. High-volume prostate patients were older, heavier and had higher BMI. Comparison adjusted for age and BMI showed higher DHT concentrations in high-volume prostate. Both normal- and high-volume prostate tissues concentrate sex steroids in a similar way. Comparison of enzymatic activity surrogate marker ratios within tissue highlighted similar TT/E1 and TT/E2 ratios, and higher DHT/E1 ratio and lower DHT/PSA ratio in the high-volume prostates. CONCLUSIONS: STERPROSER trial provides evidence for higher DHT concentration in highvolume prostates, that could reflect either higher 5-alpha reductase expression or lower expression of downstream metabolizing enzymes such as 3a-hydoxysteroid dehydrogenase.

Gleason Score within Prostate Abnormal Areas Defined by Multiparametric Magnetic Resonance Imaging Did Not Vary According to the PIRADS Score.

BACKGROUND: We aim to correlate multiparametric magnetic resonance imaging (mpMRI) of the prostate reporting (Prostate Imaging Reporting and Data System [PI-RADS] version 2) with the Gleason score into both radical prostatectomy (RP) specimen and MRI fusion-targeted biopsies (FTB). METHODS: mpMRI of 74 patients who underwent an RP after FTB were retrospectively reviewed. The Gleason score distribution was compared according to the PI-RADS score using the Kruskal-Wallis test. Results were compared to those of the mpMRI-guided biopsy of the same anatomical zone. For comparison, 903 RP specimen and their corresponding classical biopsies were also reviewed. Cohen's kappa concordance test was used to compare biopsies and prostatectomy specimen analyses. RESULTS: An exact match between Gleason grade in RP specimen and FTB was found in 62% of the cases. There was no significant difference in Gleason score ≤7 (3 + 4) vs. ≥7 (4 + 3) distribution according to the PI-RADS scores (p = 0.096). Overall, Kappa coefficients were similar with MRI-targeted biopsies compared to classical biopsies (κ = 0.378, 95% CI [0.194-0.563], and κ = 0.316, 95% CI [0.259-0.374], respectively). CONCLUSIONS: PI-RADS score was not associated with significant differences regarding Gleason score distribution within target. Moreover, concordance of Gleason score in both MRI-targeted and classical biopsies with those within target in RP specimen was weak.

A 16-gene assay to predict recurrence after surgery in localised renal cell carcinoma: development and validation studies.

BACKGROUND: The likelihood of tumour recurrence after nephrectomy in localised clear cell renal cell carcinoma is well characterised by clinical and pathological parameters. However, these assessments can be improved and personalised by the addition of molecular characteristics of each patient's tumour. We aimed to develop and validate a prognostic multigene signature to improve prediction of recurrence risk in clear cell renal cell carcinoma. METHODS: In the development stage, we investigated the association between expression of 732 genes, measured by reverse-transcription PCR, and clinical outcome in 942 patients with stage I-III clear cell renal cell carcinoma who had undergone a nephrectomy at the Cleveland Clinic (OH, USA). 516 genes were associated with recurrence-free interval. 11 of these genes were selected by further statistical analyses, and were combined with five reference genes (ie, 16 genes in total), from which a recurrence score algorithm was developed. The recurrence score was then validated in an independent cohort of 626 patients from France with stage I-III clear cell renal cell carcinoma who had also undergone nephrectomy. The association between the recurrence score and the risk of recurrence and cancer-specific survival in the first 5 years after surgery was assessed using Cox proportional hazard regression, stratified by tumour stage (stage I vs stage II vs III). FINDINGS: In our primary univariate analysis, the continuous recurrence score (median 37, IQR 31-45) was significantly associated with recurrence-free interval (hazard ratio 3·91 [95% CI 2·63-5·79] for a 25-unit increase in score, p<0·0001). In multivariable analyses, the recurrence score was significantly associated with the risk of tumour recurrence (hazard ratio per 25-unit increase in the score 3·37 [95% CI 2·23-5·08], p<0·0001) after stratification by stage and adjustment for tumour size, grade, or Leibovich score. The recurrence score was able to identify a clinically significant number of both high-risk stage I and low-risk stage II-III patients. A heterogeneity study on separate samples showed little to no intratumoural variability among the 16 genes. INTERPRETATION: Our findings validate the recurrence score as a predictor of clinical outcome in patients with stage I-III clear cell renal cell carcinoma, providing a more accurate and individualised risk assessment beyond existing clinical and pathological parameters. FUNDING: Genomic Health Inc and Pfizer Inc.

Positive surgical margins after radical prostatectomy: What should we care about?

PURPOSE: Positive surgical margins (PSMs) after radical prostatectomy (RP) are a known factor associated with biochemical recurrence (BCR) and raise the issue of adjuvant treatment by radiotherapy versus salvage treatment at recurrence. To help this choice, our study aimed to analyze BCR-free survival and factors associated with BCR in patients with PSM and undetectable postoperative prostate-specific antigen (PSA). METHODS: Between 2005 and 2008, 630 patients had RP for localized prostate cancer in our center. We included patients with PSM, uninvaded nods, undetectable postoperative PSA and no adjuvant treatment. The 5-year BCR-free survival was calculated using Kaplan-Meier method. Logistic regression models were used to determine the factors associated with BCR in univariate and multivariate analyses (Cox model). RESULTS: The PSM rate was 32.7 % (n = 206 patients), and 110 patients corresponded to the inclusion criteria. The median follow-up was 72 months. The BCR rate was 30 % with a 5-year BCR-free survival of 83.9 %. The factors significantly associated with BCR were preoperative PSA, predominance and percentage of Gleason 4, tumor volume, PSM length and predominance of Gleason 4 at the margin. In the multivariate analysis, the remaining two significant factors were PSM length [OR 4.35, 95 % CI (1.011-1.421), p = 0.037] and tumor volume [OR 4.29, 95 % CI (1.011-1.483), p = 0.038]. CONCLUSION: Over a 5-year follow-up, only one-third of patients experienced BCR. It might be reasonable to postpone adjuvant radiotherapy for patients with PSM and undetectable PSA after RP. Tumor volume and PSM length were associated with BCR and should be taken into account in the postoperative treatment management.

Intraprostatic hormone dosage: Validation of a novel prostate biopsy technique.

BACKGROUND: Advances in chromatography and mass spectrometry have allowed us to develop a novel technique for measuring intraprostatic hormone concentrations directly on prostate needle biopsies, rather than using traditional punch excision. This has significant clinical implications as intraprostatic dihydrotestosterone and testosterone levels could help monitor prostate growth, neoplasia and castration resistance. METHODS: Patients undergoing radical cystoprostatectomy for bladder cancer were prospectively included. Each prostate specimen received one 90mg punch excision and six needle biopsies. Intraprostatic hormones were dosed through gas chromatography-mass spectrometry. RESULTS: We included twenty patients, of which eleven were incidentally diagnosed with prostate cancer; four had ISUP 1 (20%) and seven had ISUP 2 (35%). The prostate biopsy technique was unable to obtain measures for testosterone, Delta-4-androsterone and androstenedione. Tissue concentrations of DHEA, DHT, E1 and E2 can be obtained with no significant difference from the reference established on a punch from a single biopsy core sample. CONCLUSIONS: Our study demonstrates that intraprostatic concentrations of DHEA, DHT, E1 and E2 can be measured without significant difference from the reference established on a single punch excision. This finding opens the way to research on the interactions between endocrinology and prostate oncogenesis and particularly on the mechanisms of resistance to hormone therapies in vivo.

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.

The value of urinary prostate cancer gene 3 (PCA3) scores in predicting pathological features at radical prostatectomy.

UNLABELLED: Study Type - Diagnostic (exploratory cohort) Level of Evidence 2b What's known on the subject? and What does the study add? PCA3 scores correlate to numerous histoprognostic factors, specifically tumour volume and positive surgical margins. These results may have a clinical impact in the near future on the selection of patients eligible to undergo active surveillance and nerve-sparing surgery. OBJECTIVE: To assess correlations between Prostate CAncer gene 3 (PCA3) levels and pathological features of radical prostatectomy (RP) specimens, which define cancer aggressiveness. PATIENTS AND METHODS: After digital rectal examination (DRE), first-catch urine was collected from 160 patients with localized prostate cancer. The PCA3 score was calculated using the Gene Probe Progensa(™) assay. PCA3 scores were then correlated to the pathological features of the RP specimens. RESULTS: PCA3 scores correlated significantly with tumour volume (r= 0.34, P < 0.01). A PCA3 score of >35 was an independent predictor in a multivariate analysis of a tumour volume >0.5 mL (odds ratio [OR] 2.7, P= 0.04). It was also an independent predictor of positive surgical margins (OR 2.4, P= 0.04). Receiver-operator characteristic curves indicated PCA3 as the most accurate predictor of positive margins (area under the curve [AUC] 0.62), in addition to a positive biopsy percentage (AUC 0.52). There was also a significant difference in the mean PCA3 score between Gleason score patient groups (6 vs ≥ 7) and pathological stage groups (pT0/2 vs pT3/4). CONCLUSIONS: PCA3 scores correlate to numerous histoprognostic factors, specifically tumour volume and positive surgical margins. These results may have a clinical impact in the near future on the selection of patients eligible to undergo active surveillance and nerve-sparing surgery.

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.

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.

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.

Prognostic Impact of pT3 Subclassification in a Multicentre Cohort of Patients with Urothelial Carcinoma of the Renal Pelvicalyceal System Undergoing Radical Nephroureterectomy: A Propensity Score-weighted Analysis After Central Pathology Review.

BACKGROUND: The current pathological tumour-node-metastasis (pTNM) classification for upper tract urothelial carcinoma (UTUC) does not include any risk stratification of pT3 renal pelvicalyceal tumours. OBJECTIVE: To assess the prognostic impact of pT3 subclassification in a multicentre cohort of patients with UTUC of the renal pelvicalyceal system undergoing radical nephroureterectomy (RNU). DESIGN, SETTING, AND PARTICIPANTS: Data from all consecutive patients treated with RNU for pT3 renal pelvicalyceal UTUC at 14 French centres from 1995 to 2013 were reviewed retrospectively. INTERVENTION: A central pathology review (CPR) was used to stratify pT3 patients into those with infiltration of the renal parenchyma on a microscopic level (pT3a) versus those with infiltration of the renal parenchyma visible on gross inspection of the resection specimen and/or invasion of peripelvic fat (pT3b). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Inverse probability weighting (IPW)-adjusted Cox regression analyses were used to compare recurrence-free survival (RFS) and cancer-specific survival (CSS) between pT3a and pT3b patients. RESULTS AND LIMITATIONS: Overall, 202 patients were included and further stratified into pT3a (n = 98; 48.5%) and pT3b (n = 104; 51.5%) subgroups. Median time to follow-up in the weighted population was 68 (interquartile range, 50-95) mo. In IPW-adjusted Cox regression analyses, pT3b versus pT3a substage was associated with a significant adverse effect on RFS (hazard ratio [HR] = 2.02; 95% confidence interval [CI] = [1.36-3.01]; p < 0.001) and CSS (HR = 1.84; 95% CI = [1.20-2.82]; p = 0.005). The study is limited by its retrospective design. CONCLUSIONS: Using IPW-adjusted analyses after the CPR, we observed that RNU patients with pT3b renal pelvicalyceal UTUC had adverse prognosis as compared with those with pT3a disease. As such, this subclassification could help refine the current pTNM system for UTUC. PATIENT SUMMARY: In this report, we looked at the prognostic interest of stratifying patients with pT3 renal pelvicalyceal upper tract urothelial carcinoma based on the extent of local invasion. We found that those with extensive infiltration (pT3b) had adverse prognosis as compared with those with limited infiltration (pT3a). This information could be provided on pathology reports to further guide clinical decision making.

Immunodynamics of explanted human tumors for immuno-oncology.

Decision making in immuno-oncology is pivotal to adapt therapy to the tumor microenvironment (TME) of the patient among the numerous options of monoclonal antibodies or small molecules. Predicting the best combinatorial regimen remains an unmet medical need. Here, we report a multiplex functional and dynamic immuno-assay based on the capacity of the TME to respond to ex vivo stimulation with twelve immunomodulators including immune checkpoint inhibitors (ICI) in 43 human primary tumors. This "in sitro" (in situ/in vitro) assay has the potential to predict unresponsiveness to anti-PD-1 mAbs, and to detect the most appropriate and personalized combinatorial regimen. Prospective clinical trials are awaited to validate this in sitro assay.

A comparative study of peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 in preclinical prostate tumour models.

INTRODUCTION: Peptide-based imaging agents targeting prostate-specific membrane antigen (PSMA) have revolutionized the evaluation of biochemical recurrence of prostate cancer (PCa) but lacks sensitivity at very low serum prostate specific antigen (PSA) levels. Once recurrence is suspected, other positron emission tomography (PET) radiotracers could be of interest to discriminate between local and distant relapse. We studied [18F]fluorodeoxyglucose ([18F]FDG) targeting glucose metabolism, [18F]fluorocholine ([18F]FCH) targeting membrane metabolism and peptide-based imaging agents [68Ga]Ga-PSMA-11, [68Ga]Ga-AMBA, [68Ga]Ga-NODAGA-RGD and [68Ga]Ga-DOTA-NT-20.3 targeting PSMA, gastrin releasing peptide receptor (GRPr), αvß3 integrin and neurotensin type 1 receptor (NTSR1) respectively, in different PCa tumour models. METHODS: Mice were xenografted with 22Rv1, an androgen-receptor (AR)-positive, PCa cell line that expresses PSMA and PC3, an AR-negative one that does not express PSMA. PET imaging using the different radiotracers was performed sequentially and the uptake characteristics compared to one other. NTSR1 and PSMA expression levels were analysed in tumours by immunohistochemistry. RESULTS: [18F]FDG displayed low but sufficient uptake to visualize PC3 and 22Rv1 derived tumours. We also observed a low efficacy of [18F]FCH PET imaging and a low [68Ga]Ga-NODAGA-RGD tumour uptake in those tumours. As expected, an elevated tumour uptake was obtained for [68Ga]Ga-PSMA-11 in 22Rv1 derived tumour although no uptake was measured in the androgen independent cell line PC3, derived from a bone metastasis of a high-grade PCa. Moreover, in PC3 cell line, we obtained good tumour uptake, high tumour-to-background contrast using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3. Immunohistochemistry analysis confirmed high NTSR1 expression in PC3 derived tumours and conversely high PSMA expression in 22Rv1 derived tumours. CONCLUSION: PET imaging using [68Ga]Ga-AMBA and [68Ga]Ga-DOTA-NT-20.3 demonstrates that GRPr and NTSR1 could represent viable alternative targets for diagnostic or therapeutic applications in PCa with limited PSMA expression levels. More preclinical and clinical studies will follow to explore this potential. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT: Peptide-based imaging agents targeting PSMA represent a major progress in the evaluation of biochemical recurrence of PCa but sometimes yield false negative results in some lesions. Continuing efforts have thus been made to evaluate other radiotracers. Our preclinical results suggest that [68Ga]labelled bombesin and neurotensin analogues could serve as alternative PET radiopharmaceuticals for diagnostic or therapy in cases of PSMA-negative PCa.