NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer.

PURPOSE: The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC. MATERIALS AND METHODS: We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs. RESULTS: NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers. CONCLUSION: NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

PD-L1 as a Urine Biomarker in Renal Cell Carcinoma-A Case Series and Proof-of-Concept Study.

BACKGROUND: Renal cell carcinoma (RCC) is among the most lethal urologic malignancies once metastatic. Current treatment approaches for metastatic RCC (mRCC) involve immune checkpoint inhibitors (ICIs) that target the PD-L1/PD-1 axis. High PD-L1 expression in tumor tissue has been identified as a negative prognostic factor in RCC. However, the role of PD-L1 as a liquid biomarker has not yet been fully explored. Herein, we analyze urine levels of PD-L1 in mRCC patients before and after either ICI therapy or surgical intervention, as well as in a series of patients with treatment-naïve RCC. PATIENTS AND METHODS: The mid-stream urine of patients with mRCC (n = 4) or treatment-naïve RCC, i.e., prior to surgery from two centers (cohort I, n = 49: cohort II, n = 29) was analyzed for PD-L1 by ELISA. The results from cohort I were compared to a control group consisting of patients treated for non-malignant urologic diseases (n = 31). In the mRCC group, urine PD-L1 levels were measured before and after tumor nephrectomy (n = 1) or before and after ICI therapy (n = 3). Exosomal PD-L1 in the urine was analyzed in selected patients by immunoblotting. RESULTS: A strong decrease in urine PD-L1 levels was found after tumor nephrectomy or following systemic treatment with ICIs. In patients with treatment-naïve RCC (cohort I), urine PD-L1 levels were significantly elevated in the RCC group in comparison to the control group (median 59 pg/mL vs. 25.7 pg/mL, p = 0.011). PD-L1 urine levels were found to be elevated, in particular, in low-grade RCCs in cohorts I and II. Exosomal PD-L1 was detected in the urine of a subset of patients. CONCLUSION: In this proof-of-concept study, we show that PD-L1 can be detected in the urine of RCC patients. Urine PD-L1 levels were found to correlate with the treatment response in mRCC patients and were significantly elevated in treatment-naïve RCC patients.

Clinical Relevance of Pathological Diagnosis of Hirschsprung's Disease with Acetylcholine-Esterase Histochemistry or Calretinin Immunohistochemistry.

INTRODUCTION: Hirschsprung disease (HD) manifests as a developmental anomaly affecting the enteric nervous system, where there is an absence of ganglion cells in the lower part of the intestine. This deficiency leads to functional blockages within the intestines. HD is usually confirmed or ruled out through rectal biopsy. The identification of any ganglion cells through hematoxylin and eosin (H&E) staining rules out HD. If ganglion cells are absent, further staining with acetylcholine-esterase (AChE) histochemistry or calretinin immunohistochemistry (IHC) forms part of the standard procedure for determining a diagnosis of HD. In 2017, our Institute of Pathology at University Hospital of Heidelberg changed our HD diagnostic procedure from AChE histochemistry to calretinin IHC. In this paper, we report the impact of the diagnostic procedure change on surgical HD therapy procedures and on the clinical outcome of HD patients. METHODS: We conducted a retrospective review of the diagnostic procedures, clinical data, and postoperative progress of 29 patients who underwent surgical treatment for HD in the Department of Pediatric Surgery, University of Heidelberg, between 2012 and 2021. The patient sample was divided into two groups, each covering a treatment period of 5 years. In 2012-2016, HD diagnosis was performed exclusively using AChE histochemistry (AChE group, n = 17). In 2017-2021, HD diagnosis was performed exclusively using calretinin IHC (CR group, n = 12). RESULTS: There were no significant differences between the groups in sex distribution, weeks of gestation, birth weight, length of the aganglionic segment, or associated congenital anomalies. Almost half of the children in the AChE group, twice as many as in the CR group, required an enterostomy before transanal endorectal pull-through procedure (TERPT). In the AChE group, 4 patients (23.5%) required repeat bowel sampling to confirm the diagnosis. Compared to the AChE group, more children in the CR group suffered from constipation post TERPT. DISCUSSION: Elevated AChE expression is linked to hypertrophied extrinsic cholinergic nerve fibers in the aganglionic segment in the majority of patients with HD. The manifestation of increased AChE expression develops over time. Therefore, in neonatal patients with HD, especially those in the first 3 weeks of life, an increase in AChE reaction is not detected. Calretinin IHC reliably identifies the presence or absence of ganglion cells and offers multiple benefits over AChE histochemistry. These include the ability to perform the test on paraffin-embedded tissue sections, a straightforward staining pattern, a clear binary interpretation (negative or positive), cost-effectiveness, and utility regardless of patient age. CONCLUSIONS: The ability of calretinin IHC to diagnose HD early and time-independently prevented repeated intestinal biopsies in our patient population and allowed us to perform a one-stage TERPT in the first months of life, reducing the number of enterostomies and restoring colonic continuity early. Patients undergoing transanal pull-through under the age of 3 months require a close follow-up to detect cases with bowel movement problems.

Knock-out of dipeptidase CN2 in human proximal tubular cells disrupts dipeptide and amino acid homeostasis and para- and transcellular solute transport.

AIM: Although of potential biomedical relevance, dipeptide metabolism has hardly been studied. We found the dipeptidase carnosinase-2 (CN2) to be abundant in human proximal tubules, which regulate water and solute homeostasis. We therefore hypothesized, that CN2 has a key metabolic role, impacting proximal tubular transport function. METHODS: A knockout of the CN2 gene (CNDP2-KO) was generated in human proximal tubule cells and characterized by metabolomics, RNA-seq analysis, paracellular permeability analysis and ion transport. RESULTS: CNDP2-KO in human proximal tubule cells resulted in the accumulation of cellular dipeptides, reduction of amino acids and imbalance of related metabolic pathways, and of energy supply. RNA-seq analyses indicated altered protein metabolism and ion transport. Detailed functional studies demonstrated lower CNDP2-KO cell viability and proliferation, and altered ion and macromolecule transport via trans- and paracellular pathways. Regulatory and transport protein abundance was disturbed, either as a consequence of the metabolic imbalance or the resulting functional disequilibrium. CONCLUSION: CN2 function has a major impact on intracellular amino acid and dipeptide metabolism and is essential for key metabolic and regulatory functions of proximal tubular cells. These findings deserve in vivo analysis of the relevance of CN2 for nephron function and regulation of body homeostasis.

Weakly Supervised MRI Slice-Level Deep Learning Classification of Prostate Cancer Approximates Full Voxel- and Slice-Level Annotation: Effect of Increasing Training Set Size.

BACKGROUND: Weakly supervised learning promises reduced annotation effort while maintaining performance. PURPOSE: To compare weakly supervised training with full slice-wise annotated training of a deep convolutional classification network (CNN) for prostate cancer (PC). STUDY TYPE: Retrospective. SUBJECTS: One thousand four hundred eighty-nine consecutive institutional prostate MRI examinations from men with suspicion for PC (65 ± 8 years) between January 2015 and November 2020 were split into training (N = 794, enriched with 204 PROSTATEx examinations) and test set (N = 695). FIELD STRENGTH/SEQUENCE: 1.5 and 3T, T2-weighted turbo-spin-echo and diffusion-weighted echo-planar imaging. ASSESSMENT: Histopathological ground truth was provided by targeted and extended systematic biopsy. Reference training was performed using slice-level annotation (SLA) and compared to iterative training utilizing patient-level annotations (PLAs) with supervised feedback of CNN estimates into the next training iteration at three incremental training set sizes (N = 200, 500, 998). Model performance was assessed by comparing specificity at fixed sensitivity of 0.97 [254/262] emulating PI-RADS ≥ 3, and 0.88-0.90 [231-236/262] emulating PI-RADS ≥ 4 decisions. STATISTICAL TESTS: Receiver operating characteristic (ROC) and area under the curve (AUC) was compared using DeLong and Obuchowski test. Sensitivity and specificity were compared using McNemar test. Statistical significance threshold was P = 0.05. RESULTS: Test set (N = 695) ROC-AUC performance of SLA (trained with 200/500/998 exams) was 0.75/0.80/0.83, respectively. PLA achieved lower ROC-AUC of 0.64/0.72/0.78. Both increased performance significantly with increasing training set size. ROC-AUC for SLA at 500 exams was comparable to PLA at 998 exams (P = 0.28). ROC-AUC was significantly different between SLA and PLA at same training set sizes, however the ROC-AUC difference decreased significantly from 200 to 998 training exams. Emulating PI-RADS ≥ 3 decisions, difference between PLA specificity of 0.12 [51/433] and SLA specificity of 0.13 [55/433] became undetectable (P = 1.0) at 998 exams. Emulating PI-RADS ≥ 4 decisions, at 998 exams, SLA specificity of 0.51 [221/433] remained higher than PLA specificity at 0.39 [170/433]. However, PLA specificity at 998 exams became comparable to SLA specificity of 0.37 [159/433] at 200 exams (P = 0.70). DATA CONCLUSION: Weakly supervised training of a classification CNN using patient-level-only annotation had lower performance compared to training with slice-wise annotations, but improved significantly faster with additional training data. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 2.

Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

Donor-Derived Cell-Free DNA (dd-cfDNA) in Kidney Transplant Recipients With Indication Biopsy-Results of a Prospective Single-Center Trial.

Donor-derived cell-free DNA (dd-cfDNA) identifies allograft injury and discriminates active rejection from no rejection. In this prospective study, 106 kidney transplant recipients with 108 clinically indicated biopsies were enrolled at Heidelberg University Hospital between November 2020 and December 2022 to validate the clinical value of dd-cfDNA in a cohort of German patients. dd-cfDNA was quantified at biopsy and correlated to histopathology. Additionally, dd-cfDNA was determined on days 7, 30, and 90 post-biopsy and analyzed for potential use to monitor response to anti-rejection treatment. dd-cfDNA levels were with a median (IQR) % of 2.00 (0.48-3.20) highest in patients with ABMR, followed by 0.92 (0.19-11.25) in patients with TCMR, 0.44 (0.20-1.10) in patients with borderline changes and 0.20 (0.11-0.53) in patients with no signs of rejection. The AUC for dd-cfDNA to discriminate any type of rejection including borderline changes from no rejection was at 0.72 (95% CI 0.62-0.83). In patients receiving anti-rejection treatment, dd-cfDNA levels significantly decreased during the 7, 30, and 90 days follow-up compared to levels at the time of biopsy (p = 0.006, p = 0.002, and p < 0.001, respectively). In conclusion, dd-cfDNA significantly discriminates active rejection from no rejection. Decreasing dd-cfDNA following anti-rejection treatment may indicate response to therapy. Clinical Trial Registration: https://drks.de/search/de/trial/DRKS00023604, identifier DRKS00023604.

Human peritoneal tight junction, transporter and channel expression in health and kidney failure, and associated solute transport.

Next to the skin, the peritoneum is the largest human organ, essentially involved in abdominal health and disease states, but information on peritoneal paracellular tight junctions and transcellular channels and transporters relative to peritoneal transmembrane transport is scant. We studied their peritoneal localization and quantity by immunohistochemistry and confocal microscopy in health, in chronic kidney disease (CKD) and on peritoneal dialysis (PD), with the latter allowing for functional characterizations, in a total of 93 individuals (0-75 years). Claudin-1 to -5, and -15, zonula occludens-1, occludin and tricellulin, SGLT1, PiT1/SLC20A1 and ENaC were consistently detected in mesothelial and arteriolar endothelial cells, with age dependent differences for mesothelial claudin-1 and arteriolar claudin-2/3. In CKD mesothelial claudin-1 and arteriolar claudin-2 and -3 were more abundant. Peritonea from PD patients exhibited increased mesothelial and arteriolar claudin-1 and mesothelial claudin-2 abundance and reduced mesothelial and arteriolar claudin-3 and arteriolar ENaC. Transperitoneal creatinine and glucose transport correlated with pore forming arteriolar claudin-2 and mesothelial claudin-4/-15, and creatinine transport with mesothelial sodium/phosphate cotransporter PiT1/SLC20A1. In multivariable analysis, claudin-2 independently predicted the peritoneal transport rates. In conclusion, tight junction, transcellular transporter and channel proteins are consistently expressed in peritoneal mesothelial and endothelial cells with minor variations across age groups, specific modifications by CKD and PD and distinct associations with transperitoneal creatinine and glucose transport rates. The latter deserve experimental studies to demonstrate mechanistic links.Clinical Trial registration: The study was performed according to the Declaration of Helsinki and is registered at www.clinicaltrials.gov (NCT01893710).

Digital Spatial Profiling Identifies the Tumor Periphery as a Highly Active Biological Niche in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by a high degree of intratumoral heterogeneity (ITH). Besides genomic ITH, there is considerable functional ITH, which encompasses spatial niches with distinct proliferative and signaling activities. The full extent of functional spatial heterogeneity in ccRCC is incompletely understood. In the present study, a total of 17 ccRCC tissue specimens from different sites (primary tumor, n = 11; local recurrence, n = 1; distant metastasis, n = 5) were analyzed using digital spatial profiling (DSP) of protein expression. A total of 128 regions of interest from the tumor periphery and tumor center were analyzed for the expression of 46 proteins, comprising three major signaling pathways as well as immune cell markers. Results were correlated to clinico-pathological variables. The differential expression of granzyme B was validated using conventional immunohistochemistry and was correlated to the cancer-specific patient survival. We found that a total of 37 proteins were differentially expressed between the tumor periphery and tumor center. Thirty-five of the proteins were upregulated in the tumor periphery compared to the center. These included proteins involved in cell proliferation, MAPK and PI3K/AKT signaling, apoptosis regulation, epithelial-to-mesenchymal transition, as well as immune cell markers. Among the most significantly upregulated proteins in the tumor periphery was granzyme B. Granzyme B upregulation in the tumor periphery correlated with a significantly reduced cancer-specific patient survival. In conclusion, this study highlights the unique cellular contexture of the tumor periphery in ccRCC. The correlation between granzyme B upregulation in the tumor periphery and patient survival suggests local selection pressure for aggressive tumor growth and disease progression. Our results underscore the potential of spatial biology for biomarker discovery in ccRCC and cancer in general.

Deep Learning-Enabled Diagnosis of Liver Adenocarcinoma.

BACKGROUND & AIMS: Diagnosis of adenocarcinoma in the liver is a frequent scenario in routine pathology and has a critical impact on clinical decision making. However, rendering a correct diagnosis can be challenging, and often requires the integration of clinical, radiologic, and immunohistochemical information. We present a deep learning model (HEPNET) to distinguish intrahepatic cholangiocarcinoma from colorectal liver metastasis, as the most frequent primary and secondary forms of liver adenocarcinoma, with clinical grade accuracy using H&E-stained whole-slide images. METHODS: HEPNET was trained on 714,589 image tiles from 456 patients who were randomly selected in a stratified manner from a pool of 571 patients who underwent surgical resection or biopsy at Heidelberg University Hospital. Model performance was evaluated on a hold-out internal test set comprising 115 patients and externally validated on 159 patients recruited at Mainz University Hospital. RESULTS: On the hold-out internal test set, HEPNET achieved an area under the receiver operating characteristic curve of 0.994 (95% CI, 0.989-1.000) and an accuracy of 96.522% (95% CI, 94.521%-98.694%) at the patient level. Validation on the external test set yielded an area under the receiver operating characteristic curve of 0.997 (95% CI, 0.995-1.000), corresponding to an accuracy of 98.113% (95% CI, 96.907%-100.000%). HEPNET surpassed the performance of 6 pathology experts with different levels of experience in a reader study of 50 patients (P = .0005), boosted the performance of resident pathologists to the level of senior pathologists, and reduced potential downstream analyses. CONCLUSIONS: We provided a ready-to-use tool with clinical grade performance that may facilitate routine pathology by rendering a definitive diagnosis and guiding ancillary testing. The incorporation of HEPNET into pathology laboratories may optimize the diagnostic workflow, complemented by test-related labor and cost savings.

Application of a validated prostate MRI deep learning system to independent same-vendor multi-institutional data: demonstration of transferability.

OBJECTIVES: To evaluate a fully automatic deep learning system to detect and segment clinically significant prostate cancer (csPCa) on same-vendor prostate MRI from two different institutions not contributing to training of the system. MATERIALS AND METHODS: In this retrospective study, a previously bi-institutionally validated deep learning system (UNETM) was applied to bi-parametric prostate MRI data from one external institution (A), a PI-RADS distribution-matched internal cohort (B), and a csPCa stratified subset of single-institution external public challenge data (C). csPCa was defined as ISUP Grade Group ≥ 2 determined from combined targeted and extended systematic MRI/transrectal US-fusion biopsy. Performance of UNETM was evaluated by comparing ROC AUC and specificity at typical PI-RADS sensitivity levels. Lesion-level analysis between UNETM segmentations and radiologist-delineated segmentations was performed using Dice coefficient, free-response operating characteristic (FROC), and weighted alternative (waFROC). The influence of using different diffusion sequences was analyzed in cohort A. RESULTS: In 250/250/140 exams in cohorts A/B/C, differences in ROC AUC were insignificant with 0.80 (95% CI: 0.74-0.85)/0.87 (95% CI: 0.83-0.92)/0.82 (95% CI: 0.75-0.89). At sensitivities of 95% and 90%, UNETM achieved specificity of 30%/50% in A, 44%/71% in B, and 43%/49% in C, respectively. Dice coefficient of UNETM and radiologist-delineated lesions was 0.36 in A and 0.49 in B. The waFROC AUC was 0.67 (95% CI: 0.60-0.83) in A and 0.7 (95% CI: 0.64-0.78) in B. UNETM performed marginally better on readout-segmented than on single-shot echo-planar-imaging. CONCLUSION: For same-vendor examinations, deep learning provided comparable discrimination of csPCa and non-csPCa lesions and examinations between local and two independent external data sets, demonstrating the applicability of the system to institutions not participating in model training. CLINICAL RELEVANCE STATEMENT: A previously bi-institutionally validated fully automatic deep learning system maintained acceptable exam-level diagnostic performance in two independent external data sets, indicating the potential of deploying AI models without retraining or fine-tuning, and corroborating evidence that AI models extract a substantial amount of transferable domain knowledge about MRI-based prostate cancer assessment. KEY POINTS: • A previously bi-institutionally validated fully automatic deep learning system maintained acceptable exam-level diagnostic performance in two independent external data sets. • Lesion detection performance and segmentation congruence was similar on the institutional and an external data set, as measured by the weighted alternative FROC AUC and Dice coefficient. • Although the system generalized to two external institutions without re-training, achieving expected sensitivity and specificity levels using the deep learning system requires probability thresholds to be adjusted, underlining the importance of institution-specific calibration and quality control.

Five-year follow-up of a phase I trial of donor-derived modified immune cell infusion in kidney transplantation.

Background: The administration of modified immune cells (MIC) before kidney transplantation led to specific immunosuppression against the allogeneic donor and a significant increase in regulatory B lymphocytes. We wondered how this approach affected the continued clinical course of these patients. Methods: Ten patients from a phase I clinical trial who had received MIC infusions prior to kidney transplantation were retrospectively compared to 15 matched standard-risk recipients. Follow-up was until year five after surgery. Results: The 10 MIC patients had an excellent clinical course with stable kidney graft function, no donor-specific human leukocyte antigen antibodies (DSA) or acute rejections, and no opportunistic infections. In comparison, a retrospectively matched control group receiving standard immunosuppressive therapy had a higher frequency of DSA (log rank P = 0.046) and more opportunistic infections (log rank P = 0.033). Importantly, MIC patients, and in particular the four patients who had received the highest cell number 7 days before surgery and received low immunosuppression during follow-up, continued to show a lack of anti-donor T lymphocyte reactivity in vitro and high CD19+CD24hiCD38hi transitional and CD19+CD24hiCD27+ memory B lymphocytes until year five after surgery. Conclusions: MIC infusions together with reduced conventional immunosuppression were associated with good graft function during five years of follow-up, no de novo DSA development and no opportunistic infections. In the future, MIC infusions might contribute to graft protection while reducing the side effects of immunosuppressive therapy. However, this approach needs further validation in direct comparison with prospective controls. Trial registration: https://clinicaltrials.gov/, identifier NCT02560220 (for the TOL-1 Study). EudraCT Number: 2014-002086-30.

Accurate tumor purity determination is critical for the analysis of homologous recombination deficiency (HRD).

Homologous recombination deficiency (HRD) is a predictive marker for response to poly (ADP-ribose) polymerase inhibitors (PARPi) in ovarian carcinoma. HRD scores have entered routine diagnostics, but the influence of algorithms, parameters and confounders has not been analyzed comprehensively. A series of 100 poorly differentiated ovarian carcinoma samples was analyzed using whole exome sequencing (WES) and genotyping. Tumor purity was determined using conventional pathology, digital pathology, and two bioinformatic methods. HRD scores were calculated from copy number profiles determined by Sequenza and by Sclust either with or without fixed tumor purity. Tumor purity determination by digital pathology combined with a tumory purity informed variant of Sequenza served as reference method for HRD scoring. Seven tumors had deleterious mutations in BRCA1/2, 12 tumors had deleterious mutations in other homologous recombination repair (HRR) genes, 18 tumors had variants of unknown significance (VUS) in BRCA1/2 or other HRR genes, while the remaining 63 tumors had no relevant alterations. Using the reference method for HRD scoring, 68 tumors were HRD-positive. HRDsum determined by WES correlated strongly with HRDsum determined by single nucleotide polymorphism (SNP) arrays (R = 0.85). Conventional pathology systematically overestimated tumor purity by 8% compared to digital pathology. All investigated methods agreed on classifying the deleterious BRCA1/2-mutated tumors as HRD-positive, but discrepancies were observed for some of the remaining tumors. Discordant HRD classification of 11% of the tumors was observed comparing the tumor purity uninformed default of Sequenza and the reference method. In conclusion, tumor purity is a critical factor for the determination of HRD scores. Assistance by digital pathology helps to improve accuracy and imprecision of its estimation.

Correction: Gupta et al. Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease. Nutrients 2022, 14, 2965.

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Carnosinase-1 Knock-Out Reduces Kidney Fibrosis in Type-1 Diabetic Mice on High Fat Diet.

Carnosine and anserine supplementation markedLy reduce diabetic nephropathy in rodents. The mode of nephroprotective action of both dipeptides in diabetes, via local protection or improved systemic glucose homeostasis, is uncertain. Global carnosinase-1 knockout mice (Cndp1-KO) and wild-type littermates (WT) on a normal diet (ND) and high fat diet (HFD) (n = 10/group), with streptozocin (STZ)-induced type-1 diabetes (n = 21-23/group), were studied for 32 weeks. Independent of diet, Cndp1-KO mice had 2- to 10-fold higher kidney anserine and carnosine concentrations than WT mice, but otherwise a similar kidney metabolome; heart, liver, muscle and serum anserine and carnosine concentrations were not different. Diabetic Cndp1-KO mice did not differ from diabetic WT mice in energy intake, body weight gain, blood glucose, HbA1c, insulin and glucose tolerance with both diets, whereas the diabetes-related increase in kidney advanced glycation end-product and 4-hydroxynonenal concentrations was prevented in the KO mice. Tubular protein accumulation was lower in diabetic ND and HFD Cndp1-KO mice, interstitial inflammation and fibrosis were lower in diabetic HFD Cndp1-KO mice compared to diabetic WT mice. Fatalities occurred later in diabetic ND Cndp1-KO mice versus WT littermates. Independent of systemic glucose homeostasis, increased kidney anserine and carnosine concentrations reduce local glycation and oxidative stress in type-1 diabetic mice, and mitigate interstitial nephropathy in type-1 diabetic mice on HFD.

Lethality of SARS-CoV-2 infection-a comparative autopsy study focusing on COVID-19 development and virus variants.

AIMS: Different SARS-CoV-2 variants are driving various waves of infection of the corona pandemic. Official statistics provide no information on who died due to coronavirus disease 2019 (COVID-19) or an alternative disease during which SARS-CoV-2 infection was detected. The current study aims at addressing the effect of the different variants evolving during the pandemic on fatal outcomes. METHODS AND RESULTS: Standardised autopsies were performed on 117 people who died of a SARS-CoV-2 infection and the findings were interpreted in clinical and pathophysiological contexts. The typical histological sequence of COVID-19-related lung injury was detected independently of the disease-causing virus variant, but was significantly less common (50 versus 80-100%) and less severe in cases infected by omicron variants compared to precedent variants (P < 0.05). COVID-19 was less often the leading cause of death following omicron infection. Extrapulmonary manifestations of COVID-19 did not contribute to death in this cohort. Lethal COVID-19 may occur after complete SARS-CoV-2 vaccination. Reinfection was not the cause of death in any of the autopsies of this cohort. CONCLUSION: Autopsies represent the gold standard in determining the cause of death after SARS-CoV-2 infection and autopsy registers are currently the only available data source allowing for evaluation of which patients died of COVID-19 or with SARS-CoV-2 infection. Compared to previous variants, infection with an omicron variant affected the lungs less frequently and resulted in less severe lung disease.

Detection Rate of Prostate Cancer in Repeat Biopsy after an Initial Negative Magnetic Resonance Imaging/Ultrasound-Guided Biopsy.

A negative multiparametric magnetic resonance imaging (mpMRI)-guided prostate biopsy in patients with suspected prostate cancer (PC) results in clinical uncertainty, as the biopsy can be false negative. The clinical challenge is to determine the optimal follow-up and to select patients who will benefit from repeat biopsy. In this study, we evaluated the rate of significant PC (sPC, Gleason score ≥7) and PC detection in patients who received a follow-up mpMRI/ultrasound-guided biopsy for persistent PC suspicion after a negative mpMRI/ultrasound-guided biopsy. We identified 58 patients at our institution that underwent repeat targeted biopsy in case of PI-RADS lesions and systematic saturation biopsy between 2014 and 2022. At the initial biopsy, the median age was 59 years, and the median prostate specific antigen level was 6.7 ng/mL. Repeat biopsy after a median of 18 months detected sPC in 3/58 (5%) patients and Gleason score 6 PC in 11/58 (19%). Among 19 patients with a downgraded PI-RADS score at the follow-up mpMRI, none had sPC. In conclusion, men with an initial negative mpMRI/ultrasound-guided biopsy had a high likelihood of not harboring sPC at repeat biopsy (95%). Due to the small size of the study, further research is recommended.

Peritoneal transformation shortly after kidney transplantation in pediatric patients with preceding chronic peritoneal dialysis.

BACKGROUND: The unphysiological composition of peritoneal dialysis (PD) fluids induces progressive peritoneal fibrosis, hypervascularization and vasculopathy. Information on these alterations after kidney transplantation (KTx) is scant. METHODS: Parietal peritoneal tissues were obtained from 81 pediatric patients with chronic kidney disease stage 5 (CKD5), 72 children on PD with low glucose degradation product (GDP) PD fluids, and from 20 children 4-8 weeks after KTx and preceding low-GDP PD. Tissues were analyzed by digital histomorphometry and quantitative immunohistochemistry. RESULTS: While chronic PD was associated with peritoneal hypervascularization, after KTx vascularization was comparable to CKD5 level. Submesothelial CD45 counts were 40% lower compared with PD, and in multivariable analyses independently associated with microvessel density. In contrast, peritoneal mesothelial denudation, submesothelial thickness and fibrin abundance, number of activated, submesothelial fibroblasts and of mesothelial-mesenchymal transitioned cells were similar after KTx. Diffuse peritoneal podoplanin positivity was present in 40% of the transplanted patients. In subgroups matched for age, PD vintage, dialytic glucose exposure and peritonitis incidence, submesothelial hypoxia-inducible factor 1-alpha abundance and angiopoietin 1/2 ratio were lower after KTx, reflecting vessel maturation, while arteriolar and microvessel p16 and cleaved Casp3 were higher. Submesothelial mast cell count and interleukin-6 were lower, whereas transforming growth factor-beta induced pSMAD2/3 was similar as compared with children on PD. CONCLUSIONS: Peritoneal membrane damage induced with chronic administration of low-GDP PD fluids was less severe after KTx. While peritoneal microvessel density, primarily defining PD transport and ultrafiltration capacity, was normal after KTx and peritoneal inflammation less pronounced, diffuse podoplanin positivity and profibrotic activity were prevalent.

Membranous NECTIN-4 Expression Frequently Decreases during Metastatic Spread of Urothelial Carcinoma and Is Associated with Enfortumab Vedotin Resistance.

PURPOSE: The antibody-drug conjugate enfortumab vedotin (EV) releases a cytotoxic agent into tumor cells via binding to the membrane receptor NECTIN-4. EV was recently approved for patients with metastatic urothelial carcinoma (mUC) without prior assessment of the tumor receptor status as ubiquitous NECTIN-4 expression is assumed. Our objective was to determine the prevalence of membranous NECTIN-4 protein expression in primary tumors (PRIM) and patient-matched distant metastases (MET). EXPERIMENTAL DESIGN: Membranous NECTIN-4 protein expression was measured (H-score) by IHC in PRIM and corresponding MET (N = 137) and in a multicenter EV-treated cohort (N = 47). Progression-free survival (PFS) after initiation of EV treatment was assessed for the NECTIN-4-negative/weak (H-score 0-99) versus moderate/strong (H-score 100-300) subgroup. The specificity of the NECTIN-4 IHC staining protocol was validated by establishing CRISPR-Cas9-induced polyclonal NECTIN-4 knockouts. RESULTS: In our cohort, membranous NECTIN-4 expression significantly decreased during metastatic spread (Wilcoxon matched pairs P < 0.001; median H-score = 40; interquartile range, 0-140), with 39.4% of MET lacking membranous NECTIN-4 expression. In our multicenter EV cohort, absence or weak membranous NECTIN-4 expression (34.0% of the cohort) was associated with a significantly shortened PFS on EV (log-rank P < 0.001). CONCLUSIONS: Membranous NECTIN-4 expression is frequently decreased or absent in mUC tissue. Of note, the clinical benefit of EV strongly depends on membranous NECTIN-4 expression. Thus, our results are of highest clinical relevance and argue for a critical reconsideration of the current practice and suggest that the NECTIN-4 receptor status should be determined (ideally in a metastatic/progressive lesion) before initiation of EV. See related commentary by Aggen et al., p. 1377.