Therapeutic and Diagnostic Potential of Folic Acid Receptors and Glycosylphosphatidylinositol (GPI) Transamidase in Prostate Cancer.

Due to the proliferation-induced high demand of cancer cells for folic acid (FA), significant overexpression of folate receptors 1 (FR1) is detected in most cancers. To our knowledge, a detailed characterization of FR1 expression and regulation regarding therapeutic and diagnostic feasibilities in prostate cancer (PCa) has not been described. In the present study, cell cultures, as well as tissue sections, were analyzed using Western blot, qRT-PCR and immunofluorescence. In addition, we utilized FA-functionalized lipoplexes to characterize the potential of FR1-targeted delivery into PCa cells. Interestingly, we detected a high level of FR1-mRNA in healthy prostate epithelial cells and healthy prostate tissue. However, we were able to show that PCa cells in vitro and PCa tissue showed a massively enhanced FR1 membrane localization where the receptor can finally gain its function. We were able to link these changes to the overexpression of GPI-transamidase (GPI-T) by image analysis. PCa cells in vitro and PCa tissue show the strongest overexpression of GPI-T and thereby induce FR1 membrane localization. Finally, we utilized FA-functionalized lipoplexes to selectively transfer pDNA into PCa cells and demonstrate the therapeutic potential of FR1. Thus, FR1 represents a very promising candidate for targeted therapeutic transfer pathways in PCa and in combination with GPI-T, may provide predictive imaging in addition to established diagnostics.

Dual role of GRHL3 in bladder carcinogenesis depending on histological subtypes.

The effect of grainyhead-like transcription factor 3 (GRHL3) on cancer development depends on the cancer subtypes as shown in tumor entities such as colorectal or oral squamous cell carcinomas. Here, we analyzed the subtype-specific role of GRHL3 in bladder carcinogenesis, comparing common urothelial carcinoma (UC) with squamous bladder cancer (sq-BLCA). We examined GRHL3 mRNA and protein expression in cohorts of patient samples, its prognostic role and its functional impact on tumorigeneses in different molecular and histopathological subtypes of bladder cancer. We showed for GRHL3 a reverse expression in squamous and urothelial bladder cancer subtypes. Stably GRHL3-overexpressing EJ28, J82, and SCaBER in vitro models revealed a tumor-suppressive function in squamous and an oncogenic role in the urothelial cancer cells affecting cell and colony growth, and migratory and invasive capacities. Transcriptomic profiling demonstrated highly subtype-specific GRHL3-regulated expression networks coined by the enrichment of genes involved in integrin-mediated pathways. In SCaBER, loss of ras homolog family member A (RHOA) GTPase activity was demonstrated to be associated with co-regulation of eukaryotic translation initiation factor 4E family member 3 (EIF4E3), a potential tumor suppressor gene. Thus, our data provide for the first time a detailed insight into the role of the transcription factor GRHL3 in different histopathological subtypes of bladder cancer.

Alpha-1 Antitrypsin Augmentation and the Liver Phenotype of Adults With Alpha-1 Antitrypsin Deficiency (Genotype Pi∗ZZ).

BACKGROUND & AIMS: α1-Antitrypsin (AAT) is a major protease inhibitor produced by hepatocytes. The most relevant AAT mutation giving rise to AAT deficiency (AATD), the 'Pi∗Z' variant, causes harmful AAT protein accumulation in the liver, shortage of AAT in the systemic circulation, and thereby predisposes to liver and lung injury. Although intravenous AAT augmentation constitutes an established treatment of AATD-associated lung disease, its impact on the liver is unknown. METHODS: Liver-related parameters were assessed in a multinational cohort of 760 adults with severe AATD (Pi∗ZZ genotype) and available liver phenotyping, of whom 344 received augmentation therapy and 416 did not. Liver fibrosis was evaluated noninvasively via the serum test AST-to-platelet ratio index and via transient elastography-based liver stiffness measurement. Histologic parameters were compared in 15 Pi∗ZZ adults with and 35 without augmentation. RESULTS: Compared with nonaugmented subjects, augmented Pi∗ZZ individuals displayed lower serum liver enzyme levels (AST 71% vs 75% upper limit of normal, P < .001; bilirubin 49% vs 58% upper limit of normal, P = .019) and lower surrogate markers of fibrosis (AST-to-platelet ratio index 0.34 vs 0.38, P < .001; liver stiffness measurement 6.5 vs 7.2 kPa, P = .005). Among biopsied participants, augmented individuals had less pronounced liver fibrosis and less inflammatory foci but no differences in AAT accumulation were noted. CONCLUSIONS: The first evaluation of AAT augmentation on the Pi∗ZZ-related liver disease indicates liver safety of a widely used treatment for AATD-associated lung disease. Prospective studies are needed to confirm the beneficial effects and to demonstrate the potential efficacy of exogenous AAT in patients with Pi∗ZZ-associated liver disease.

An international multi-institutional validation study of the algorithm for prostate cancer detection and Gleason grading.

Pathologic examination of prostate biopsies is time consuming due to the large number of slides per case. In this retrospective study, we validate a deep learning-based classifier for prostate cancer (PCA) detection and Gleason grading (AI tool) in biopsy samples. Five external cohorts of patients with multifocal prostate biopsy were analyzed from high-volume pathology institutes. A total of 5922 H&E sections representing 7473 biopsy cores from 423 patient cases (digitized using three scanners) were assessed concerning tumor detection. Two tumor-bearing datasets (core n = 227 and 159) were graded by an international group of pathologists including expert urologic pathologists (n = 11) to validate the Gleason grading classifier. The sensitivity, specificity, and NPV for the detection of tumor-bearing biopsies was in a range of 0.971-1.000, 0.875-0.976, and 0.988-1.000, respectively, across the different test cohorts. In several biopsy slides tumor tissue was correctly detected by the AI tool that was initially missed by pathologists. Most false positive misclassifications represented lesions suspicious for carcinoma or cancer mimickers. The quadratically weighted kappa levels for Gleason grading agreement for single pathologists was 0.62-0.80 (0.77 for AI tool) and 0.64-0.76 (0.72 for AI tool) for the two grading datasets, respectively. In cases where consensus for grading was reached among pathologists, kappa levels for AI tool were 0.903 and 0.855. The PCA detection classifier showed high accuracy for PCA detection in biopsy cases during external validation, independent of the institute and scanner used. High levels of agreement for Gleason grading were indistinguishable between experienced genitourinary pathologists and the AI tool.

Scoring Systems for Immunohistochemistry in Urothelial Carcinoma.

Immunohistochemistry is widely used in diagnostic and scientific analysis of urothelial carcinoma. Objective interpretation of staining results is mandatory for accuracy and comparability in diagnostic and therapeutic patient care as well as research.Herein we summarize and explain standardized microscopic evaluation and scoring approaches for immunohistochemical stainings. We focus on commonly used and generally feasible approaches for different cellular compartments and comment on their utility in diagnostics and research practice.

Genetic polymorphisms in interleukin-1ß (rs1143634) and interleukin-8 (rs4073) are associated with survival after resection of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a rare, understudied primary hepatic malignancy with dismal outcomes. Aiming to identify prognostically relevant single-nucleotide polymorphisms, we analyzed 11 genetic variants with a role in tumor-promoting inflammation (VEGF, EGF, EGFR, IL-1B, IL-6, CXCL8 (IL-8), IL-10, CXCR1, HIF1A and PTGS2 (COX-2) genes) and their association with disease-free (DFS) and overall survival (OS) in patients undergoing curative-intent surgery for iCCA. Genomic DNA was isolated from 112 patients (64 female, 48 male) with iCCA. Germline polymorphisms were analyzed with polymerase chain reaction-restriction fragment length polymorphism protocols. The IL-1B +3954 C/C (73/112, hazard ratio (HR) = 1.735, p = 0.012) and the IL-8 -251 T/A or A/A (53/112 and 16/112, HR = 2.001 and 1.1777, p = 0.026) genotypes were associated with shorter OS in univariable and multivariable analysis. The IL-1B +3954 polymorphism was also associated with shorter DFS (HR = 1.983, p = 0.012), but this effect was not sustained in the multivariable model. A genetic risk model of 0, 1 and 2 unfavorable alleles was established and confirmed in multivariable analysis. This study supports the prognostic role of the IL-1B C+3954T and the IL-8 T-251A variant as outcome markers in iCCA patients, identifying patient subgroups at higher risk for dismal clinical outcomes.

[Urothelial carcinoma of the upper urinary tract]. / Urothelkarzinom des oberen Harntrakts.

Urothelial carcinoma of the upper urinary tract (upper tract urothelial carcinoma, UTUC) is less common than bladder carcinoma with nearly identical risk factors and has a poorer prognosis. The standard diagnostic procedure is imaging of the upper urinary tract by computed tomography urography. In cases of diagnostic uncertainty, a diagnostic ureterorenoscopy with biopsy sampling can be performed in addition to urine cytology. Treatment depends primarily on the stage and grading of the tumor. Depending on the extent and localization, organ-preserving treatment or radical nephroureterectomy is indicated. Perioperative systemic treatment in high-risk UTUC can be performed in both neoadjuvant and adjuvant settings, although the current data on neoadjuvant chemo- and immunotherapy do not yet allow standard application. For metastatic disease, a multimodal treatment approach consisting of cisplatin-based or carboplatin-based chemotherapy, immunotherapy, and treatment with enfortumab vedotin can be considered. Salvage surgery, radiotherapy and metastasectomy are available for rare individual cases.

Mechanisms of organ fibrosis: Emerging concepts and implications for novel treatment strategies.

Fibrosis, or tissue scarring, develops as a pathological deviation from the physiological wound healing response and can occur in various organs such as the heart, lung, liver, kidney, skin, and bone marrow. Organ fibrosis significantly contributes to global morbidity and mortality. A broad spectrum of etiologies can cause fibrosis, including acute and chronic ischemia, hypertension, chronic viral infection (e.g., viral hepatitis), environmental exposure (e.g., pneumoconiosis, alcohol, nutrition, smoking) and genetic diseases (e.g., cystic fibrosis, alpha-1-antitrypsin deficiency). Common mechanisms across organs and disease etiologies involve a sustained injury to parenchymal cells that triggers a wound healing response, which becomes deregulated in the disease process. A transformation of resting fibroblasts into myofibroblasts with excessive extracellular matrix production constitutes the hallmark of disease, however, multiple other cell types such as immune cells, predominantly monocytes/macrophages, endothelial cells, and parenchymal cells form a complex network of profibrotic cellular crosstalk. Across organs, leading mediators include growth factors like transforming growth factor-ß and platelet-derived growth factor, cytokines like interleukin-10, interleukin-13, interleukin-17, and danger-associated molecular patterns. More recently, insights into fibrosis regression and resolution of chronic conditions have deepened our understanding of beneficial, protective effects of immune cells, soluble mediators and intracellular signaling. Further in-depth insights into the mechanisms of fibrogenesis can provide the rationale for therapeutic interventions and the development of targeted antifibrotic agents. This review gives insight into shared responses and cellular mechanisms across organs and etiologies, aiming to paint a comprehensive picture of fibrotic diseases in both experimental settings and in human pathology.

Optical coherence tomography combined with convolutional neural networks can differentiate between intrahepatic cholangiocarcinoma and liver parenchyma ex vivo.

PURPOSE: Surgical resection with complete tumor excision (R0) provides the best chance of long-term survival for patients with intrahepatic cholangiocarcinoma (iCCA). A non-invasive imaging technology, which could provide quick intraoperative assessment of resection margins, as an adjunct to histological examination, is optical coherence tomography (OCT). In this study, we investigated the ability of OCT combined with convolutional neural networks (CNN), to differentiate iCCA from normal liver parenchyma ex vivo. METHODS: Consecutive adult patients undergoing elective liver resections for iCCA between June 2020 and April 2021 (n = 11) were included in this study. Areas of interest from resection specimens were scanned ex vivo, before formalin fixation, using a table-top OCT device at 1310 nm wavelength. Scanned areas were marked and histologically examined, providing a diagnosis for each scan. An Xception CNN was trained, validated, and tested in matching OCT scans to their corresponding histological diagnoses, through a 5 × 5 stratified cross-validation process. RESULTS: Twenty-four three-dimensional scans (corresponding to approx. 85,603 individual) from ten patients were included in the analysis. In 5 × 5 cross-validation, the model achieved a mean F1-score, sensitivity, and specificity of 0.94, 0.94, and 0.93, respectively. CONCLUSION: Optical coherence tomography combined with CNN can differentiate iCCA from liver parenchyma ex vivo. Further studies are necessary to expand on these results and lead to innovative in vivo OCT applications, such as intraoperative or endoscopic scanning.

Direct prediction of Homologous Recombination Deficiency from routine histology in ten different tumor types with attention-based Multiple Instance Learning: a development and validation study.

Background: Homologous Recombination Deficiency (HRD) is a pan-cancer predictive biomarker that identifies patients who benefit from therapy with PARP inhibitors (PARPi). However, testing for HRD is highly complex. Here, we investigated whether Deep Learning can predict HRD status solely based on routine Hematoxylin & Eosin (H&E) histology images in ten cancer types. Methods: We developed a fully automated deep learning pipeline with attention-weighted multiple instance learning (attMIL) to predict HRD status from histology images. A combined genomic scar HRD score, which integrated loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large-scale state transitions (LST) was calculated from whole genome sequencing data for n=4,565 patients from two independent cohorts. The primary statistical endpoint was the Area Under the Receiver Operating Characteristic curve (AUROC) for the prediction of genomic scar HRD with a clinically used cutoff value. Results: We found that HRD status is predictable in tumors of the endometrium, pancreas and lung, reaching cross-validated AUROCs of 0.79, 0.58 and 0.66. Predictions generalized well to an external cohort with AUROCs of 0.93, 0.81 and 0.73 respectively. Additionally, an HRD classifier trained on breast cancer yielded an AUROC of 0.78 in internal validation and was able to predict HRD in endometrial, prostate and pancreatic cancer with AUROCs of 0.87, 0.84 and 0.67 indicating a shared HRD-like phenotype is across tumor entities. Conclusion: In this study, we show that HRD is directly predictable from H&E slides using attMIL within and across ten different tumor types.

Abbreviated MRI for Comprehensive Regional Lymph Node Staging during Pre-Operative Breast MRI.

BACKGROUND: The detection of regional lymph node metastases (LNM), in particular significant LNM (≥N2), is important to guide treatment decisions in women with breast cancer. The purpose of this study was to determine whether a coronal pulse sequence as part of pre-operative breast MRI is useful to identify women without significant LNM. MATERIAL: Retrospective study between January 2017 and December 2019 on 414 consecutive women with breast cancer who underwent pre-operative breast MRI on a 1.5 T system. For lymph node (LN) staging, a coronal pre-contrast non-fat-suppressed T1-weighted TSE sequence was acquired with the system's built-in body coil, covering the chest wall; acquisition time 3:12 min. Two radiologists rated the likelihood of LNM on a 3-point scale (absent/possible/present). Validation was obtained by histology from sentinel LN biopsy, axillary LN dissection, and/or PET/CT. RESULTS: 368/414 women were staged to have no or non-significant LNM (pN0 in 282/414, pN1 in 86/414), and significant LNM (≥pN2) in 46/414. For identification of women with significant LNM, MRI was true-positive in 42/46, false-negative in 4/46, true-negative in 327/368, and false-positive in 41/83, the latter mostly caused by women with N1-disease (38/41), yielding an NPV and PPV for significant LNM of 98.8% [95%-CI: 97.0-100%] and 50.6% [43.1-58.1%], respectively. CONCLUSIONS: A 3 min coronal T1-weighted pulse sequence covering the chest wall as part of pre-operative breast MRI is useful to rule out significant LNM with high NPV. Where MRI staging is positive for significant LNM, additional work-up is indicated to improve the distinction of N1 and N2 disease.

Characterisation of tumour-immune phenotypes and PD-L1 positivity in squamous bladder cancer.

AIMS: Immune checkpoint inhibitor (ICI) therapy has become a viable treatment strategy in bladder cancer. However, treatment responses vary, and improved biomarkers are needed. Crucially, the characteristics of immune cells remain understudied especially in squamous differentiated bladder cancer (sq-BLCA). Here, we quantitatively analysed the tumour-immune phenotypes of sq-BLCA and correlated them with PD-L1 expression and FGFR3 mutation status. METHODS: Tissue microarrays (TMA) of n = 68 non-schistosomiasis associated pure squamous cell carcinoma (SCC) and n = 46 mixed urothelial carcinoma with squamous differentiation (MIX) were subjected to immunohistochemistry for CD3, CD4, CD8, CD56, CD68, CD79A, CD163, Ki67, perforin and chloroacetate esterase staining. Quantitative image evaluation was performed via digital image analysis. RESULTS: Immune infiltration was generally higher in stroma than in tumour regions. B-cells (CD79A) were almost exclusively found in stromal areas (sTILs), T-lymphocytes and macrophages were also present in tumour cell areas (iTILs), while natural killer cells (CD56) were nearly missing in any area. Tumour-immune phenotype distribution differed depending on the immune cell subset, however, hot tumour-immune phenotypes (high density of immune cells in tumour areas) were frequently found for CD8 + T-cells (33%), especially perforin + lymphocytes (52.2%), and CD68 + macrophages (37.6%). Perforin + CD8 lymphocytes predicted improved overall survival in sq-BLCA while high PD-L1 expression (CPS ≥ 10) was significantly associated with higher CD3 + , CD8 + and CD163 + immune cell density and high Ki67 (density) of tumour cells. Furthermore, PD-L1 expression was positively associated with CD3 + /CD4 + , CD3 + /CD8 + and CD68 + /CD163 + hot tumour-immune phenotypes. FGFR3 mutation status was inversely associated with CD8 + , perforin + and CD79A + lymphocyte density. CONCLUSIONS: Computer-based image analysis is an efficient tool to analyse immune topographies in squamous bladder cancer. Hot tumour-immune phenotypes with strong PD-L1 expression might pose a promising subgroup for clinically successful ICI therapy in squamous bladder cancer and warrant further investigation.

[New WHO classification 2022: urinary bladder cancer]. / Neue WHO-Klassifikation 2022: Harnblasenkarzinom.

The new World Health Organization (WHO) classification of urogenital tumors is still primarily based on anatomic location, but is also a hierarchical taxonomic classification without separate chapters for tumors of the upper urinary tract and the urethra. It clarifies aspects regarding grading and noninvasive entities. It consolidates the use of the Paris system for urinary cytology as well as various subtypes/special types of neoplasms, and incorporates general concepts of the 5th edition of the WHO blue book. In addition to mesenchymal tumors, well-differentiated neuroendocrine tumors and neuroendocrine carcinomas are addressed in separate chapters. Papillary non-invasive low- and high-grade carcinomas and carcinoma in situ remain, while dysplasia and urothelial proliferation of unknown malignant potential (UPUMP) are no longer treated as separate entities. Former variants of urothelial carcinoma are now called subtypes and aberrant differentiation and special types are more precisely defined.

Clinical sequencing identifies potential actionable alterations in a high rate of urachal and primary bladder adenocarcinomas.

OBJECTIVE: Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. METHODS: Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions. RESULTS: After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients. CONCLUSIONS: In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments.

Effective Radiosensitization of Bladder Cancer Cells by Pharmacological Inhibition of DNA-PK and ATR.

This study aims at analyzing the impact of the pharmacological inhibition of DNA damage response (DDR) targets (DNA-PK and ATR) on radiosensitization of bladder cancer cell lines of different molecular/histological subtypes. Applying DNA-PK (AZD7648) and ATR (Ceralasertib) inhibitors on SCaBER, J82 and VMCUB-1 bladder cancer cell lines, we revealed sensitization upon ionizing radiation (IR), i.e., the IC50 for each drug shifted to a lower drug concentration with increased IR doses. In line with this, drug exposure retarded DNA repair after IR-induced DNA damage visualized by a neutral comet assay. Western blot analyses confirmed specific inhibition of targeted DDR pathways in the analyzed bladder cancer cell lines, i.e., drugs blocked DNA-PK phosphorylation at Ser2056 and the ATR downstream mediator CHK1 at Ser317. Interestingly, clonogenic survival assays indicated a cell-line-dependent synergism of combined DDR inhibition upon IR. Calculating combined index (CI) values, with and without IR, according to the Chou-Talalay method, confirmed drug- and IR-dose-specific synergistic CI values. Thus, we provide functional evidence that DNA-PK and ATR inhibitors specifically target corresponding DDR pathways retarding the DNA repair process at nano-molar concentrations. This, in turn, leads to a strong radiosensitizing effect and impairs the survival of bladder cancer cells.

Genetic Variant of CXCR1 (rs2234671) Associates with Clinical Outcome in Perihilar Cholangiocarcinoma.

Background: Perihilar cholangiocarcinoma (pCCA) is a rare primary liver malignancy. Even in patients amenable to surgery, outcomes are often dismal. Here, we aimed to identify prognostic markers for patient outcomes by analyzing functionally relevant single-nucleotide polymorphisms (SNPs) in genes with a role in tumor inflammation and angiogenesis. We analyzed 11 polymorphisms in the inflammation-angiogenesis axis (VEGF, EGF, EGFR, IL-1b, IL-6, CXCL8 (IL-8), IL-10, CXCR1, HIF1A, and COX2 genes) for their prediction of tumor recurrence and survival in pCCA patients undergoing surgery in a curative intent. Methods: Samples were obtained from 111 patients with pCCA undergoing liver resection in curative intent. DNA was extracted and analyzed using polymerase chain reaction-restriction fragment length polymorphism protocols and correlated with patients' outcomes. Results: Out of the assessed variants, only the CXCR1 (also: interleukin-8-receptor alpha - IL-8RA) +860C>G heterozygous polymorphism (rs2234671) was associated with decreased disease-free survival (DFS), cancer-specific survival (CSS), and overall survival (OS) (18/111 (16.2%), median DFS 14 months, log-rank p = 0.007; median CSS 31 months, log-rank p = 0.007; and median OS 6 months, log-rank p = 0.002), compared to the GG genotype (92/111 (82.9%), median DFS 55 months, median CSS 63 months, and median OS 33 months). In the multivariate analysis, +860C>G remained an independent prognostic factor for DFS (adjusted p = 0.008), CSS (adjusted p = 0.001), and OS (adjusted p = 0.001). Conclusion: Genetic variant of CXCR1 +860C>G may serve as a molecular marker for DFS, CSS, and OS in patients undergoing curative-intent surgery for pCCA, indicating that the analysis of SNPs in genes involved in immune-mediated angiogenesis may help to identify patient subgroups at high risk for dismal oncological and overall outcome.