The contribution of new methods in cytology for increasing sensitivity in the diagnosis of extrahepatic bile duct lesions.

The aim of this review is to provide a comprehensive analysis of the existing literature pertaining to cytology of extrahepatic bile ducts. A search using the keywords "biliary brush cytology" was conducted in the PubMed database, with a focus on recent articles. The inclusion criteria primarily encompassed publications addressing problematic biliary stenosis. Emphasis was placed on identifying articles that explored innovative or less-utilized examination techniques aimed at enhancing the sensitivity of cytological examination. This review presents a comprehensive overview of the various types of materials used in sampling and the corresponding sampling methods. Additionally, it explores cytological and cytogenetic techniques, such as fluorescence in situ hybridization (FISH) and genetic methods (miRNA, NGS, cfDNA). These techniques possess the potential to improve the accuracy of diagnosing bile duct tumors, although their sensitivity varies. Furthermore, their utilization can facilitate early therapy, which plays a crucial role in patient prognosis. Each examination is always dependent on the quality and quantity of material delivered. A higher sensitivity of these examinations can be achieved by combining biliary cytology and other complementary methods.

Fluorescence In Situ Hybridization in Primary Diagnosis of Biliary Strictures: A Single-Center Prospective Interventional Study.

Background and aims: Diagnosis of the biliary stricture remains a challenge. In view of the low sensitivity of brush cytology (BC), fluorescence in situ hybridization (FISH) has been reported as a useful adjunctive test in patients with biliary strictures. We aimed to determine performance characteristics of BC and FISH individually and in combination (BC + FISH) in the primary diagnosis of biliary strictures. Methods: This single-center prospective study was conducted between April 2019 and January 2021. Consecutive patients with unsampled biliary strictures undergoing first endoscopic retrograde cholangiopancreatography in our institution were included. Tissue specimens from two standardized transpapillary brushings from the strictures were examined by routine cytology and FISH. Histopathological confirmation after surgery or 12-month follow-up was regarded as the reference standard for final diagnosis. Results: Of 109 enrolled patients, six were excluded and one lost from the final analysis. In the remaining 102 patients (60.8% males, mean age 67.4, range 25-92 years), the proportions of benign and malignant strictures were 28 (27.5%) and 74 (72.5%), respectively. The proportions of proximal and distal strictures were 26 (25.5%) and 76 (74.5%), respectively. In comparison to BC alone, FISH increased the sensitivity from 36.1% to 50.7% (p = 0.076) while maintaining similar specificity (p = 0.311). Conclusions: Dual-modality tissue evaluation using BC + FISH showed an improving trend in sensitivity for the primary diagnosis of biliary strictures when compared with BC alone.

A-ring-fused pyrazoles of dihydrotestosterone targeting prostate cancer cells via the downregulation of the androgen receptor.

High expression of the androgen receptor (AR) and the disruption of its regulation are strongly responsible for the development of prostate cancer (PCa). Therapeutically relevant non-steroidal or steroidal antiandrogens are able to block the AR effect by eliminating AR-mediated signalling. Herein we report the synthesis of novel steroidal pyrazoles derived from the natural sex hormone 5α-dihydrotestosterone (DHT). 2-Ethylidene or 2-(hetero)arylidene derivatives of DHT obtained by regioselective Claisen-Schmidt condensation with acetaldehyde or (hetero)aromatic aldehydes in alkaline ethanol were reacted with monosubstituted hydrazines to give A-ring-fused 1,5-disubstituted pyrazoles as main or exclusive products, depending on the reaction conditions applied. Spontaneous or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)-induced oxidation of the primarily formed pyrazolines resulted in the desired products in moderate to good yields, while 17-oxidation also occurred by using the Jones reagent as a strong oxidant. Transcriptional activity of the AR in a reporter cell line was examined for all novel compounds, and several previously synthesized similar DHT-based pyrazoles with differently substituted heteroring were also included to obtain information about the structure-activity relationship. Two specific regioisomeric groups of derivatives significantly diminished the transcriptional activity of the AR in reporter cell line in 10 µM concentration, and displayed reasonable antiproliferative activity in AR-positive PCa cell lines. Lead compound (3d) was found to be a potent AR antagonist (IC50 = 1.18 µM), it generally suppressed AR signalling in time and dose dependent manner, moreover, it also led to a sharp decrease in wt-AR protein level probably caused by proteasomal degradation. We confirmed the antiproliferative activity of 3d in AR-positive PCa cell lines (with GI50 in low micromolar ranges), and its cellular, biochemical and in silico binding in AR ligand-binding domain. Moreover, compound 3d was shown to be potent even ex vivo in patient-derived tissues, which highlights the therapeutic potential of A-ring-fused pyrazoles.

Modulating the pharmacokinetic profile of Actinium-225-labeled macropa-derived radioconjugates by dual targeting of PSMA and albumin.

Rationale: Small 225Ac-labeled prostate-specific membrane antigen (PSMA)-targeted radioconjugates have been described for targeted alpha therapy of metastatic castration-resistant prostate cancer. Transient binding to serum albumin as a highly abundant, inherent transport protein represents a commonly applied strategy to modulate the tissue distribution profile of such low-molecular-weight radiotherapeutics and to enhance radioactivity uptake into tumor lesions with the ultimate objective of improved therapeutic outcome. Methods: Two ligands mcp-M-alb-PSMA and mcp-D-alb-PSMA were synthesized by combining a macropa-derived chelator with either one or two lysine-ureido-glutamate-based PSMA- and 4-(p-iodophenyl)butyrate albumin-binding entities using multistep peptide-coupling chemistry. Both compounds were labeled with [225Ac]Ac3+ under mild conditions and their reversible binding to serum albumin was analyzed by an ultrafiltration assay as well as microscale thermophoresis measurements. Saturation binding studies and clonogenic survival assays using PSMA-expressing LNCaP cells were performed to evaluate PSMA-mediated cell binding and to assess the cytotoxic potency of the novel radioconjugates [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Biodistributions of both 225Ac-radioconjugates were investigated using LNCaP tumor-bearing SCID mice. Histological examinations of selected organs were performed to analyze the occurrence of necrosis using H&E staining, DNA damage via γH2AX staining and proliferation via Ki67 expression in the tissue samples. Results: Enhanced binding to serum components in general and to human serum albumin in particular was revealed for [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Moreover, the novel derivatives are highly potent PSMA ligands as their KD values in the nanomolar range (23.38 and 11.56 nM) are comparable to the reference radioconjugates [225Ac]Ac-mcp-M-PSMA (30.83 nM) and [225Ac]Ac-mcp-D-PSMA (10.20 nM) without albumin binders. The clonogenic activity of LNCaP cells after treatment with the 225Ac-labeled ligands was affected in a dose- and time-dependent manner, whereas the bivalent radioconjugate [225Ac]Ac-mcp-D-alb-PSMA has a stronger impact on the clonogenic cell survival than its monovalent counterpart [225Ac]Ac-mcp-M-alb-PSMA. Biodistribution studies performed in LNCaP tumor xenografts showed prolonged blood circulation times for both albumin-binding radioconjugates and a substantially increased tumor uptake (46.04 ± 7.77 %ID/g for [225Ac]Ac-mcp-M-alb-PSMA at 128 h p.i. and 153.48 ± 37.76 %ID/g at 168 h p.i. for [225Ac]Ac-mcp-D-alb-PSMA) with favorable tumor-to-background ratios. Consequently, a clear histological indication of DNA damage was discovered in the tumor tissues, whereas DNA double-strand break formation in kidney and liver sections was less pronounced. Conclusion: The modification of the PSMA-based 225Ac-radioconjugates with one or two albumin-binding entities resulted in an improved radiopharmacological behavior including a greatly enhanced tumor accumulation combined with a rather low uptake in most non-targeted organs combined with a high excretion via the kidneys.

Iodinated Contrast Medium Affects Urine Cytology Assessment: A Prospective, Single-Blind Study and Its Impact on Urological Practice.

During endoscopic procedures for suspected urothelial tumors of the upper urinary tract, radiographic imaging using an iodinated contrast medium is often required. However, following ureteropyelography, we detected changes in cytology characteristics not correlating with real cytology findings in naive urine. The aim of our study was to assess cytology changes between naive and postcontrast urine according to The Paris System of cytology classification. Methods: We prospectively assessed urine samples from 89 patients (23 patients with histologically proven urothelial cancer and 66 healthy volunteers). The absence of malignancy was demonstrated by CT urography and/or ureteroscopy. The study was single blind (expert cytopathologist) and naïve Paris system for urine cytology assessment was used. Furthermore, additional cytological parameters were analyzed (e.g., specimen cellularity, degree of cytolysis, cytoplasm and nucleus color, chromatin and nucleo-cytoplasmic ratio). Results: Our study showed statistically significant differences when comparing naïve and postcontrast urine in healthy volunteers (only 51 % concordance, p = 0.001) versus malignant urine specimens (82 % concordance). The most important differences were in the shift from The Paris System category 2 (negative) to 1 (non-diagnostic) and from category 2 (negative) to 3 (atypia). Other significant changes were found in the assessment of specimen cellularity (p = 0.0003), degree of cytolysis (p = 0.001), cytoplasm color (p = 0.003), hyperchromasia (p = 0.001), course chromatin (p = 0.002), nucleo-cytoplasmatic ratio (p = 0.001) and nuclear borders' irregularity (p = 0.01). Conclusion: Our unique study found crucial changes in the cytological assessment of naive and postcontrast urine and we confirm that postcontrast urine is more often assessed as abnormal, suspect or non-diagnostic. Therefore, before urine collection for cytology, the clinician should avoid administration of iodinated contrast into the urinary tract.

Toll-Like Receptor 3 Overexpression Induces Invasion of Prostate Cancer Cells, whereas Its Activation Triggers Apoptosis.

Toll-like receptor 3 (TLR3) is an endosomal receptor expressed in several immune and epithelial cells. Recent studies have highlighted its expression also in solid tumors, including prostate cancer (PCa), and have described its role primarily in the proinflammatory response and induction of apoptosis. It is up-regulated in some castration-resistant prostate cancers. However, the role of TLR3 in prostate cancer progression remains largely unknown. The current study experimentally demonstrated that exogenous TLR3 activation in PCa cell lines leads to a significant induction of secretion of the cytokines IL-6, IL-8, and interferon-ß, depending on the model and chemoresistance status. Transcriptomic analysis of TLR3-overexpressing cells revealed a functional program that is enriched for genes involved in the regulation of cell motility, migration, and tumor invasiveness. Increased motility, migration, and invasion in TLR3-overexpressing cell line were confirmed by several in vitro assays and using an orthotopic prostate xenograft model in vivo. Furthermore, TLR3-ligand induced apoptosis via cleavage of caspase-3/7 and poly (ADP-ribose) polymerase, predominantly in TLR3-overexpressing cells. These results indicate that TLR3 may be involved in prostate cancer progression and metastasis; however, it might also represent an Achilles heel of PCa, which can be exploited for targeted therapy.

Surgical treatment of duodenal adenocarcinoma: ampullary vs. non-ampullary, short- and long-term outcomes.

BACKGROUND: The aim of this study was to evaluate symptoms, diagnostic methods, short- and long-term outcomes of surgical treatment in patients with duodenal adenocarcinoma. PATIENTS AND METHODS: A single center, retrospective, observational study of 52 consecutive patients with duodenal adenocarcinoma operated on with curative intent between 2006 - 2019. Duodenectomy as part of a hemipancreatoduodenectomy or total pancreatectomy procedure was performed for ADAC (ampullary duodenal/intestinal adenocarcinoma) or NADAC (non-ampullary duodenal adenocarcinoma). RESULTS: Prevailing symptoms were obstructive jaundice in the ADAC group (P<0.0001) and bleeding in the NADAC group (P=0.005), with larger tumor size in patients with NADAC (P=0.001). Complication rate, morbidity and mortality were comparable. Primary total pancreatoduodenectomy predominated in the NADAC group, 16.6% vs. 2.9%, and salvage completion pancreatectomy in the ADAC group, 6% vs. 0%. Significant prognostic factors for OS were perineural invasion (P=0.006) and adjuvant chemotherapy (P=0.045) in the ADAC group, and for DFS the total number of resected lymph nodes (P=0.042) and lymph node ratio (P=0.031) in the NADAC group. Median OS is 21 months and 5-year survival 27.3% in the NADAC group and 41.5 months and 52% in the ADAC group. CONCLUSION: Ampullary duodenal/intestinal adenocarcinomas are smaller than non-ampullary at diagnosis, with a higher rate of lymph node metastases, but with a better prognosis and long-term outcome in the presented cohort. Oral localisation of NADAC prevailed in the present cohort. Perineural invasion and postoperative oncological therapy are significant prognostic factors for OS in ADAC, but the total number of lymph nodes and lymph node ratio are significant prognostic factors for DFS in NADAC.

Skp2 and Slug Are Coexpressed in Aggressive Prostate Cancer and Inhibited by Neddylation Blockade.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.

Do not underestimate anterior prostate cancer.

AIMS: With the introduction of magnetic resonance imaging in the diagnosis of prostate cancer and its use in targeted prostate biopsy, an increased incidence of anterior-predominant prostate cancer (APC) has been observed. METHODS: We enrolled 200 patients who underwent radical prostatectomy at our department between 12/2017 and 04/2019. We evaluated tumour location in the individual segments of the prostate, index tumour location and volume, and compared the postoperative stage, Gleason score, grade group (GG), and the presence of extraprostatic extension (EPE) in APC and posterior prostate cancer (PPC). We assessed the rate of MRI scans prior to prostate surgery as well as the influence of family history and PSA on the presence of APC. RESULTS: We found a significantly higher rate of anterior tumours than previously reported (37%) and confirmed that these tumours are diagnosed with a significantly larger index tumour volume (P=0.003). We also showed that a mere 6.76% of APCs were low-risk tumours not requiring radical treatment. Furthermore, anterior tumours were found significantly more often (P=0.001) in patients who underwent preoperative MRI. No differences were observed between PSA values, family history, presence of EPE, or locally advanced disease in APC vs. PPC. CONCLUSIONS: The frequency of anterior tumours is higher than previously thought, and they include tumours requiring radical treatment. When these tumours are neglected, it may lead to patient undertreatment with impact on their life prognosis. Thus, we consider the use of MRI-targeted prostate biopsy to be a necessity both for ruling out APC in the case of repeatedly negative prostate biopsies and, in particular, before patient inclusion in active surveillance.

Targeting genotoxic and proteotoxic stress-response pathways in human prostate cancer by clinically available PARP inhibitors, vorinostat and disulfiram.

BACKGROUND: Castration-resistant prostate cancer (PCa) represents a serious health challenge. Based on mechanistically-supported rationale we explored new therapeutic options based on clinically available drugs with anticancer effects, including inhibitors of PARP1 enzyme (PARPi), and histone deacetylases (vorinostat), respectively, and disulfiram (DSF, known as alcohol-abuse drug Antabuse) and its copper-chelating metabolite CuET that inhibit protein turnover. METHODS: Drugs and their combination with ionizing radiation (IR) were tested in various cytotoxicity assays in three human PCa cell lines including radio-resistant stem-cell like derived cells. Mechanistically, DNA damage repair, heat shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and immunoblotting. RESULTS: We observed enhanced sensitivity to PARPi/IR in PC3 cells consistent with lower homologous recombination (HR) repair. Vorinostat sensitized DU145 cells to PARPi/IR and decreased mutant p53. Vorinostat also impaired HR-mediated DNA repair, as determined by Rad51 foci formation and downregulation of TOPBP1 protein, and overcame radio-resistance of stem-cell like DU145-derived cells. All PCa models responded well to CuET or DSF combined with copper. We demonstrated that DSF interacts with copper in the culture media and forms adequate levels of CuET indicating that DSF/copper and CuET may be considered as comparable treatments. Both DSF/copper and CuET evoked hallmarks of UPR in PCa cells, documented by upregulation of ATF4, CHOP and phospho-eIF2α, with ensuing heat shock response encompassing activation of HSF1 and HSP70. Further enhancing the cytotoxicity of CuET, combination with an inhibitor of the anti-apoptotic protein survivin (YM155, currently undergoing clinical trials) promoted the UPR-induced toxicity, yielding synergistic effects of CuET and YM155. CONCLUSIONS: We propose that targeting genotoxic and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.

Generation of human iPSCs from human prostate cancer-associated fibroblasts IBPi002-A.

A human induced pluripotent stem cell line was generated from cancer-associated fibroblasts of a 68-years old patient with diagnosed prostate adenocarcinoma (PCa). The fibroblast cell line was reprogrammed with Epi5™ Episomal iPSC Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting expression of factors of pluripotency on a single-cell level, and also in vivo using teratoma formation assay. This new iPS cell line may be used for differentiation into different prostate-specific cell types in differentiation studies.

Presence of growth/differentiation factor-15 cytokine in human follicular fluid, granulosa cells, and oocytes.

PURPOSE: The purpose of the study was to determine whether the GDF-15 is present in follicular fluid; to evaluate if there is a relation between follicular and serum levels of GDF-15 and fertility status of study subjects; and to test whether granulosa cells, oocytes, or both produce GDF-15. METHODS: This study used follicular fluid (FF, serum, and oocytes obtained under informed consent from women undergoing oocyte retrieval for in vitro fertilization. It also used ovaries from deceased preterm newborns. Collection of FF and blood at the time of oocyte retrieval, ELISA and western blot were performed to determine levels and forms of GDF-15. Concentrations of GDF-15 in FF and serum, its expression in ovarian tissue, and secretion from granulosa cells were analyzed. RESULTS: GDF-15 concentration in FF ranged from 35 to 572 ng/ml, as determined by ELISA. Western blot analysis revealed the GDF-15 pro-dimer only in FF. Both normal healthy and cancerous granulosa cells secreted GDF-15 into culture media. Primary oocytes displayed cytoplasmic GDF-15 positivity in immunostained newborn ovaries, and its expression was also observed in fully grown human oocytes. CONCLUSIONS: To the best of our knowledge, this is the first documentation of cytokine GDF-15 presence in follicular fluid. Its concentration was not associated with donor/patient fertility status. Our data also show that GDF-15 is expressed and inducible in both normal healthy and cancerous granulosa cells, as well as in oocytes.

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-ß1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry.

DNA damage signalling barrier, oxidative stress and treatment-relevant DNA repair factor alterations during progression of human prostate cancer.

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP.