Recent developments in the diagnosis and management of N1 penile cancer.

PURPOSE OF REVIEW: This article presents a critical review of the current literature to provide a brief update on the contemporary advances in diagnosing and managing N1 penile cancer. RECENT FINDINGS: Penile squamous cell carcinoma (pSCC) has evolved from being an orphan field for cancer innovation. Advances in the understanding tumour biology have enabled sophisticated diagnostics and predictive modelling to better characterize inguinal disease. Minimally invasive inguinal lymph node dissection is emerging as a technique that reduces morbidity while maintaining oncological safety. Furthermore, robust clinical trials are underway ,which will provide level one evidence to guide treatment decisions. Exciting advances in the field of immune-oncology offer promise as adjuvant therapies. International collaboration and centralisation of care will be essential to driving translational research and equitable evidence-based care. SUMMARY: Improving outcomes for men with pSCC remains a global challenge. Radical inguinal lymph node dissection remains the gold standard for diagnosing and curing N1 disease. Although many promising developments are on the horizon, high-level evidence is required to guide therapy.

Fully Automated Protein Proximity Assay in Formalin-Fixed, Paraffin-Embedded Tissue Using Caged Haptens.

The ability to interrogate for the presence and distribution of protein-protein complexes (PPCs) is of high importance for the advancement of diagnostic capabilities such as determining therapeutic effects in the context of pharmaceutical development. Herein, we report a novel assay for detecting and visualizing PPCs on formalin-fixed, paraffin-embedded material using a caged hapten. To this end, we synthetically modified a nitropyrazole hapten with an alkaline phosphatase (AP)-responsive self-immolative caging group. The AP-labile caging group abrogates antibody binding; however, upon exposure to AP, the native hapten is regenerated. These caged haptens were applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the population of PPCs, was then visualized by an anti-hapten antibody conjugated to horseradish peroxidase, followed by diaminobenzidine detection. We provide proof of concept for the detection of protein proximity pairs (ß-catenin-E-cadherin and EGFR-GRB2), and confirm assay specificity through technical controls involving reagent omission experiments, and biologically by treatment with small-molecule kinase inhibitors that interrupt kinase-adaptor complexes.

Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker.

PEGylation typically improves the systemic exposure and tumor biodistribution of polymeric drug delivery systems, but may also restrict enzyme access to peptide-based drug linkers. The impact of dendrimer generation (G4 vs G5) and PEG length (570 vs 1100 Da) on the pharmacokinetics, tumor biodistribution, drug release kinetics, and anticancer activity of a series of PEGylated polylysine dendrimers conjugated with doxorubicin via a cathepsin-B cleavable valine-citrulline linker was therefore investigated in rodents. Although the smallest G4 PEG570 dendrimer showed the most efficient cathepsin-mediated doxorubicin release, systemic exposure and tumor uptake were limited. The largest G5 PEG1100 dendrimer showed good tumor uptake and retention but restricted drug liberation and therefore limited anticancer activity. Superior anticancer activity was achieved using an intermediate sized dendrimer that showed better drug release kinetics, systemic exposure, tumor uptake, and retention. The data suggest that balancing PEG molecular weight and dendrimer size is critical when designing chemotherapeutic dendrimers.

Lymphatic transport and lymph node targeting of methotrexate-conjugated PEGylated dendrimers are enhanced by reducing the length of the drug linker or masking interactions with the injection site.

Drug conjugation to dendrimer-based delivery systems has been shown to enhance delivery to the lymphatic system after subcutaneous administration. Dendrimer interaction with components of the interstitium at the injection site, however, may prevent drainage from the injection site. The current study sought to vary the length of a linker employed to conjugate methotrexate (MTX) to a PEGylated dendrimer, in an attempt to reduce MTX interaction with interstitial binding sites and enhance lymphatic drainage. Dendrimers with shorter linkers resulted in higher lymphatic drainage, presumably via shielding of interaction sites by the PEG mantle, but were not retained in lymph nodes. Improved drainage of dendrimers with longer linkers was achieved through coadministration with dextran to mask interactions at the injection site while maintaining retention within the node. Enhanced drug exposure to the lymph node has the potential to enhance the treatment of lymph-node resident cancer metastases.

Quinone Methide Signal Amplification: Covalent Reporter Labeling of Cancer Epitopes using Alkaline Phosphatase Substrates.

Diagnostic assays with the sensitivity required to improve cancer therapeutics depend on the development of new signal amplification technologies. Herein, we report the development and application of a novel amplification system which utilizes latent quinone methides (QMs) activated by alkaline phosphatase (AP) for signal amplification in solid-phase immunohistochemical (IHC) assays. Phosphate-protected QM precursor substrates were prepared and conjugated to either biotin or a fluorophore through an amine-functionalized linker group. Upon reaction with AP, the phosphate group is cleaved, followed by elimination of the leaving group and formation of the highly reactive and short-lived QM. The QMs either react with tissue nucleophiles in close proximity to their site of generation, or are quenched by nucleophiles in the reaction media. The reporter molecules that covalently bind to the tissue were then detected visually by fluorescence microscopy in the case of fluorophore reporters, or brightfield microscopy using diaminobenzidine (DAB) in the case of biotin reporters. With multiple reporters deposited per enzyme, significant signal amplification was observed utilizing QM precursor substrates containing either benzyl difluoro or benzyl monofluoro leaving group functionalities. However, the benzyl monofluoro leaving group gave superior results with respect to both signal intensity and discretion, the latter of which was found to be imperative for use in diagnostic IHC assays.

The assessment of HER2 status in breast cancer: the past, the present, and the future.

Humanized monoclonal anti-human growth factor receptor 2 (HER2) antibody trastuzumab was approved for HER2 positive breast cancer patient treatment 11 years after the demonstration of HER2 gene amplification associated with the HER2 protein overexpression in breast cancer in 1987. HER2 positive status of breast cancer patients is assessed by HER2 gene amplification with in situ hybridization (ISH) and/or HER2 protein overexpression with immunohistochemistry (IHC). Because the discordance between quantitative HER2 ISH and subjective, semi-quantitative HER2 IHC assay results is a well-recognized issue of HER2 testing, we developed an assay combining HER2 ISH and HER2 IHC assays (HER2 gene-protein assay; HER2 GPA) as one test on the same tissue section. HER2 GPA allows pathologists to score the HER2 gene and HER2 protein status simultaneously at the individual cell level. The possibility that HER2 GPA may become the next generation of HER2 testing is discussed, particularly for cases in which it is difficult to assess the HER2 status of breast cancer patients due to the HER2 heterogeneity.

Methotrexate-conjugated PEGylated dendrimers show differential patterns of deposition and activity in tumor-burdened lymph nodes after intravenous and subcutaneous administration in rats.

The current study sought to explore whether the subcutaneous administration of lymph targeted dendrimers, conjugated with a model chemotherapeutic (methotrexate, MTX), was able to enhance anticancer activity against lymph node metastases. The lymphatic pharmacokinetics and antitumor activity of PEGylated polylysine dendrimers conjugated to MTX [D-MTX(OH)] via a tumor-labile hexapeptide linker was examined in rats and compared to a similar system where MTX was α-carboxyl O-tert-butylated [D-MTX(OtBu)]. The latter has previously been shown to exhibit longer plasma circulation times. D-MTX(OtBu) was well absorbed from the subcutaneous injection site via the lymph, and 3 to 4%/g of the dose was retained by sentinel lymph nodes. In contrast, D-MTX(OH) showed limited absorption from the subcutaneous injection site, but absorption was almost exclusively via the lymph. The retention of D-MTX(OH) by sentinel lymph nodes was also significantly elevated (approximately 30% dose/g). MTX alone was not absorbed into the lymph. All dendrimers displayed lower lymph node targeting after intravenous administration. Despite significant differences in the lymph node retention of D-MTX(OH) and D-MTX(OtBu) after subcutaneous and intravenous administration, the growth of lymph node metastases was similarly inhibited. In contrast, the administration of MTX alone did not significantly reduce lymph node tumor growth. Subcutaneous administration of drug-conjugated dendrimers therefore provides an opportunity to improve drug deposition in downstream tumor-burdened lymph nodes. In this case, however, increased lymph node biodistribution did not correlate well with antitumor activity, possibly suggesting constrained drug release at the site of action.

The acquisition and retention of urinary catheterisation skills using surgical simulator devices: teaching method or student traits.

BACKGROUND: The acquisition of procedural skills is an essential component of learning for medical trainees. The objective of this study was to assess which teaching method of performing urinary catheterisation is associated with most efficient procedural skill acquisition and retention. We evaluated factors affecting acquisition and retention of skills when using simulators as adjuncts to medical training. METHODS: Forty-two second year medical students were taught urinary catheter insertion using different teaching methods. The interactive group (n = 19) were taught using a lecture format presentation and a high fidelity human urinary catheter simulator. They were provided with the use of simulators prior to examination. The observer group (n = 12) were taught using the same method but without with simulator use prior to examination. The didactic group (n = 11) were taught using the presentation alone. Student characteristics such as hand dexterity and IQ were measured to assess for intrinsic differences. All students were examined at four weeks to measure skill retention. RESULTS: Catheter scores were significantly higher in the interactive group (p < 0.005). Confidence scores with catheter insertion were similar at index exam however were significantly lower in the didactic group at the retention testing (p < 0.05). Retention scores were higher in the interactive group (p < 0.001). A significant positive correlation was observed between laparoscopy scores and time to completion with overall catheter score (p < 0.05). Teaching method, spatial awareness and time to completion of laparoscopy were significantly associated with higher catheter scores at index exam (p = 0.001). Retention scores at 4 weeks were significantly associated with teaching method and original catheter score (p = 0.001). CONCLUSION: The importance of simulators in teaching a complex procedural skill has been highlighted. Didactic teaching method was associated with a significantly higher rate of learning decay at retention testing.

Digital analysis of the prostate tumor microenvironment with high-order chromogenic multiplexing.

As our understanding of the tumor microenvironment grows, the pathology field is increasingly utilizing multianalyte diagnostic assays to understand important characteristics of tumor growth. In clinical settings, brightfield chromogenic assays represent the gold-standard and have developed significant trust as the first-line diagnostic method. However, conventional brightfield tests have been limited to low-order assays that are visually interrogated. We have developed a hybrid method of brightfield chromogenic multiplexing that overcomes these limitations and enables high-order multiplex assays. However, how compatible high-order brightfield multiplexed images are with advanced analytical algorithms has not been extensively evaluated. In the present study, we address this gap by developing a novel 6-marker prostate cancer assay that targets diverse aspects of the tumor microenvironment such as prostate-specific biomarkers (PSMA and p504s), immune biomarkers (CD8 and PD-L1), a prognostic biomarker (Ki-67), as well as an adjunctive diagnostic biomarker (basal cell cocktail) and apply the assay to 143 differentially graded adenocarcinoma prostate tissues. The tissues were then imaged on our spectroscopic multiplexing imaging platform and mined for proteomic and spatial features that were correlated with cancer presence and disease grade. Extracted features were used to train a UMAP model that differentiated healthy from cancerous tissue with an accuracy of 89% and identified clusters of cells based on cancer grade. For spatial analysis, cell-to-cell distances were calculated for all biomarkers and differences between healthy and adenocarcinoma tissues were studied. We report that p504s positive cells were at least 2× closer to cells expressing PD-L1, CD8, Ki-67, and basal cell in adenocarcinoma tissues relative to the healthy control tissues. These findings offer a powerful insight to understand the fingerprint of the prostate tumor microenvironment and indicate that high-order chromogenic multiplexing is compatible with digital analysis. Thus, the presented chromogenic multiplexing system combines the clinical applicability of brightfield assays with the emerging diagnostic power of high-order multiplexing in a digital pathology friendly format that is well-suited for translational studies to better understand mechanisms of tumor development and growth.

Complicated artificial urinary sphincter insertion using the transcorporal cuff: keys to the tunical flap technique.

Urethral sphincter insufficiency following radical prostatectomy (RP) is a common cause of non-neurogenic stress urinary incontinence (SUI). Artificial urinary sphincter (AUS) insertion remains the standard of care for fit patients with SUI refractory to non-operative interventions. The proximal urethra is a common location for uncomplicated AUS placement. However, previous failed AUS, urethroplasty, or pelvic radiotherapy (RT) may compromise urethral tissue requiring technique modifications that optimise outcomes. In these situations, transcorporal cuff (TC) placement has been well described to facilitate continence restoration in men where there is no other feasible option other than urinary diversion or permanent incontinence. In the traditional TC approach, the procedure may be complicated by haematoma due to difficulty in completely closing the corporal defects behind the urethra. This narrated video demonstrates the tunical flap (TF) modification for transcorporal AUS implantation via a perineal and penoscrotal approach in patients with prior failed AUS placements secondary to urethral erosion. The TF technique for transcorporal AUS insertion provides circumferential reinforcement with tunica albuginea from the corpora cavernosa. Here, we show how this technique provides additional urethral support for compromised urethral tissue to help prevent cuff erosion. The TF preserves the corporal volume and does not limit candidacy for future penile prosthesis implantation. In our early results, there have been no postoperative haematoma formation with this technique.

Pulmonary administration of PEGylated polylysine dendrimers: absorption from the lung versus retention within the lung is highly size-dependent.

The systemic delivery of drugs via the inhaled route is an attractive, needle-free means of improving the systemic exposure of molecules such as peptides and proteins that are poorly absorbed after oral administration. Directed delivery into the lungs also provides a means of increasing drug concentrations at the site of action for lung-specific disease states such as pulmonary infections and lung cancer. The current study has examined the potential utility of PEGylated polylysine dendrimers as pulmonary delivery agents and in particular sought to explore the relationship between dendrimer size and absorption of the intact construct (as a potential systemic delivery mechanism) versus retention within the lungs (as a potential pulmonary depot for controlled local release). Dendrimer absorption from the lungs was inversely correlated with molecular weight, with approximately 20-30% of the dose of relatively small (<22 kDa) dendrimers systemically absorbed compared to only 2% absorption for a larger (78 kDa) PEGylated dendrimer. Increasing the molecular weight of the dendrimers led to slower absorption and more prolonged retention in the lung tissue and bronchoalveolar lavage fluid. Oral administration of the two smaller dendrimers confirmed that oral bioavailability of the PEGylated dendrimers was essentially zero and did not contribute to exposure after pulmonary administration. The smaller PEGylated dendrimers were also degraded in the lungs to low molecular weight products that were subsequently absorbed and excreted via the urine, while the larger constructs showed good stability in the lungs. The data suggest first, that small PEGylated dendrimer-based drug delivery systems may be delivered to the blood via inhalation, providing a more attractive alternative to injections, and second that larger PEGylated dendrimers may be retained in the lungs providing the potential for controlled delivery of medications to the blood or lung tissue.

A review of expression profiling of circulating microRNAs in men with prostate cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in gene regulation and expression. These molecules have been implicated in the pathogenesis of several malignancies. MiRNAs have the potential to be both a tissue- and blood-based biomarker for cancer diagnosis, classification, prognosis and response to treatment. The levels of dysregulated miRNAs in the circulation of individuals make miRNAs an ideal biomarker for the detection of prostate cancer. The present study provides a comprehensive review of the expression profiling of miRNAs in the circulation of men with prostate cancer. Further research is necessary to identify if circulating miRNAs have the potential to be a biomarker for prostate cancer in clinical practice.

Social determinants of health: does socioeconomic status affect access to staging imaging for men with prostate cancer.

Socioeconomic status (SES), race and geographical factors are known to impact prostate cancer management and outcomes. We aimed to assess these factors with regard to access to novel imaging in prostate cancer. Using the Prostate Cancer Outcomes Registry of Victoria (PCOR-Vic) we identified 5256 men diagnosed with prostate cancer via biopsy. Following the introduction of government rebate, the access to MRI improved with respect to SES. Access to PET imaging remains poor with respect to SES and geographical location in the absence of Federal funding. Further improvements for men with low SES and regional areas to access PET staging.

Combination treatment in metastatic prostate cancer: is the bar too high or have we fallen short?

Androgen deprivation therapy (ADT) alone has been the cornerstone of treatment for patients with newly diagnosed metastatic prostate cancer for the past century. Based on results from landmark trials in the past decade, combination approaches of ADT with chemotherapy or novel hormonal agents have established a new standard of care for these patients. This paradigm shift in treatment has been reflected in the updates to guideline recommendations of major professional associations. However, real-world data from around the world have highlighted the dismal adoption of combination therapy, despite evidence-based recommendations. The disparity between evidence and practice is concerning, especially with emerging evidence of survival benefit with further treatment intensification using triplet combinations (ADT, docetaxel and novel hormonal agents). Thus, a pressing need to raise awareness and call the uro-oncology community to action exists to deliver evidence-based care for these patients.