Development of minimally invasive approaches in penile cancer

Introduction: Penile cancer (PeCa) is a rare genital malignancy. The prognosis for patients presenting early and with no involvement of the inguinal lymph nodes is excellent. Advances in penile preserving surgery and avoiding overtreatment of inguinal lymph nodes aims to reduce the morbidity of surgery. The aim of this thesis is to investigate penile cancer surgical techniques to reduce morbidity.Methods: A total of 10 peer-reviewed research papers (as senior author) were critically evaluated. These included surgical treatment for primary disease which described novel techniques and predictors for local recurrence using a consecutive cohort of patients for data analysis. The outcomes from dynamic sentinel lymph node biopsy and the management of non-visualisation were evaluated using a complete dataset of patients with impalpable inguinal lymph nodes. Finally, the management of inguinal lymph nodes using a fascial sparing technique used a data set from 2 specialist centres and combined the data to report on the surgical outcomes.Results: The results from the studies included in this thesis show that with penile preserving procedures such as glansectomy, the local recurrence rate is less than 10%. The results from the dynamic sentinel lymph node studies showed that a delayed procedure was feasible with a sensitivity of 88.9%. The non-visualisation group can be visualised on repeat dynamic sentinel node biopsy in 86% of cases. The complication rate of fascial sparing inguinal lymphadenectomy is 36.1% and lower than previously reported series. The use of novel techniques such as “Batman’ scrotectomy and myocutaneous flaps is associated with low wound complications and allows palliation of patients.Discussion: The management of penile cancer has undergone significant change over the study period. The use of minimally invasive surgery has reduced morbidity and not significantly impacted on the local recurrence rate or cancer specific survival.</p

sj-docx-2-uro-10.1177_20514158211073426 – Supplemental material for Microdissection oncoTESE (micro-oncoTESE) in azoospermic men with suspected testicular cancer: Analysis of outcomes from a specialist centre

Supplemental material, sj-docx-2-uro-10.1177_20514158211073426 for Microdissection oncoTESE (micro-oncoTESE) in azoospermic men with suspected testicular cancer: Analysis of outcomes from a specialist centre by Asheesh Kaul, Athos Katelaris, Aiman Haider, Alex Freeman, Kamran Ahmed, Hussain M Alnajjar and Asif Muneer in Journal of Clinical Urology</p

sj-docx-1-uro-10.1177_20514158211073426 – Supplemental material for Microdissection oncoTESE (micro-oncoTESE) in azoospermic men with suspected testicular cancer: Analysis of outcomes from a specialist centre

Supplemental material, sj-docx-1-uro-10.1177_20514158211073426 for Microdissection oncoTESE (micro-oncoTESE) in azoospermic men with suspected testicular cancer: Analysis of outcomes from a specialist centre by Asheesh Kaul, Athos Katelaris, Aiman Haider, Alex Freeman, Kamran Ahmed, Hussain M Alnajjar and Asif Muneer in Journal of Clinical Urology</p

Understanding the role of adenosine receptors in the myofibroblast transformation in Peyronie’s disease

Background: Peyronie’s disease (PD) is a chronic fibrotic disease of the penis affecting a significant number of men worldwide without effective medical treatments. Myofibroblasts are pivotal in the pathogenesis of PD. Adenosine and adenosine receptors have been suggested to be involved in the pathophysiology of fibrosis. Aim: To understand the role of adenosine receptors in myofibroblast transformation in PD. Methods: Fibroblasts were isolated from the non-PD tunica albuginea (TA) tissue and PD plaque tissue and were transformed into myofibroblasts using transforming growth factor β1 (TGF-β1). Quantification of alpha smooth muscle actin (α-SMA) and adenosine receptors (ADORA1, ADORA2A, ADORA2B and ADORA3) was performed using immunocytochemistry (ICC), In-Cell ELISA (ICE) and real-time RT-PCR (RT-qPCR). The effect of various adenosine receptor agonists or antagonists on TGF-β1-induced myofibroblast transformation was measured using ICE. Outcomes: Expression of adenosine receptors in myofibroblasts obtained from human TA and the effect of adenosine receptor ligands on myofibroblast transformation. Results: ICC, ICE and RT-qPCR experiments showed that the protein and mRNA levels of α-SMA in non- PD TA cells and PD plaque-derived cells were significantly higher in cells exposed to TGF-β1 than those not treated with TGF-β1. Two of four adenosine receptors (ADORA1 and ADORA2B) were found to be expressed in both cell populations. Among various adenosine receptor agonists/antagonist investigated, only ADORA2B agonist, BAY60-6583, significantly inhibited myofibroblast transformation in a concentration-dependent manner when applied simultaneously with TGF-β1 (IC50=30 μM). Clinical Translation: ADORA2B antagonists may be clinically efficacious in early stage PD. Strengths & Limitations: The strength of this study is the use of primary fibroblasts from human TA. Limitation of the study is the high concentrations of the ligands used. Conclusions: The effect of an ADORA2B agonist on TGF-β1-induced myofibroblast transformation shows a novel potential therapeutic target for PD if applied during early, non-stable phase of PD

Semen sampling as a simple, noninvasive surrogate for prostate health screening

The detection rates for prostate cancer (pCa) by invasive biopsy are high, fully justifying its use in confirmatory testing. False-positive results of prior, relatively insensitive screening tests, however, can lead to expensive and often unnecessary surgery. Several reports have suggested the potential use of the ejaculate to screen for prostate conditions. Hitherto, the potential impact of sterilization on the diagnostic potential of seminal plasma screening has not been examined. Herein, we report cellular and molecular comparisons of semen samples obtained from normal (N = 5), vasectomized (N = 5) and prostate pathology patients (N = 4; confirmed by a biopsy) that were centrifuged over 60% PureSperm cushions. Non-penetrating cells were washed prior to immunocytochemistry with prostatic epithelial cell markers including PSMA, NKX3.1 and CD24. KRT18 was used to highlight epithelial cells in these samples. RNA sequencing was then used to identify differentially expressed small RNAs associated with vasectomy and prostate pathology. Specific gene transcripts were confirmed by RT-qPCR. PMSA+/KRT18+, CD24+/KRT18+ and NKX3.1/+KRT18+ cells were observed, albeit infrequently in most processed semen samples by indirect immunocytochemistry. Targeted RT-qPCR supported their enrichment, along with their putative designation as prostatic luminal cells. Small RNAs in seminal plasma were highly heterogeneous, with tRNAs and miRNAs being the dominant forms. Hsa-miR-143 and hsa-miR-199 were among the most prominent of the differentially expressed miRNAs upregulated in samples with prostate pathology but not vasectomy. The targets of these small RNAs illustrate biological processes involved among others in transcription regulation and collagen metabolism. Our outcomes strongly support an appraisal of selected biologically meaningful small RNAs of ejaculate semen for prostate health screening. A long-term goal would be a simple, routine, noninvasive test for monitoring prostate health, potentially among younger men.</p

Phosphodiesterase Type 5 Inhibitors and Selective Estrogen Receptor Modulators Can Prevent But Not Reverse Myofibroblast Transformation in Peyronie's Disease

Background: Myofibroblast transformation is a key step in the pathogenesis of Peyronie's disease (PD). Phosphodiesterase type 5 inhibitors (PDE5is) and selective estrogen receptor modulators (SERMs) can prevent the formation of fibrosis in in vitro and in vivo models of PD. However, it is unknown whether these drugs can also reverse established fibrosis. Aim: To investigate whether PDE5is and SERMs can reverse transforming growth factor beta 1 (TGF-β1)–induced myofibroblast transformation and determine the point of no return. Methods: In-Cell enzyme-linked immunosorbent assay was used to quantify TGF-β1–induced myofibroblast transformation of human primary fibroblasts isolated from tunica albuginea (TA) of patients undergoing surgery for treatment of PD. Extracellular matrix production and collagen contraction assays were used as secondary assays. Reverse transcription–quantitative polymerase chain reaction and In-Cell enzyme-linked immunosorbent assay were used to measure drug target expression. PDE5i (vardenafil) and SERM (tamoxifen) were applied at various time points after TGF-β1. Outcomes: Reversibility of myofibroblast transformation and drug target expression were investigated in a time-dependent manner in TA-derived fibroblasts. Results: Vardenafil or tamoxifen could not reverse the myofibroblast traits of alpha-smooth muscle actin expression and extracellular matrix production, whereas only tamoxifen affected collagen contraction after 72 hours of TGF-β1 treatment. Phosphodiesterase 5A and estrogen receptor (ER)-β were downregulated after 72 hours, and estrogen receptor -α protein could not be quantified. Tamoxifen could prevent myofibroblast transformation until 36 hours after TGF-β1 treatment, whereas vardenafil could prevent only 24 hours after TGF-β1 treatment. This was mirrored by downregulation of drug targets on mRNA and protein level. Furthermore, antifibrotic signaling pathways, peroxisome proliferator-activated receptor gamma and betaglycan (TGFB receptor III), were significantly downregulated after 36 hours of TGF-β1 exposure, as opposed to upregulation of profibrotic thrombospondin-1 at the same time point. Clinical Translation: This study suggests that using PDE5is and SERMs might only help for early-phase PD and further highlights the need to test drugs at the appropriate stage of the disease based on their mechanism of action. Strengths & Limitations: The study uses primary human TA-derived fibroblasts that enhances translatability of the results. Limitations include that only 1 example of PDE5i- and SERM-type drug was tested. Time course experiments were only performed for marker expression experiments and not for functional assays. Conclusion: This is the first study to demonstrate that timing for administration of drugs affecting myofibroblast transformation appears to be vital in in vitro models of PD, where 36 hours of TGF-β1 treatment can be suggested as a “point of no return” for myofibroblast transformation

Antifibrotic synergy between phosphodiesterase type 5 inhibitors and selective oestrogen receptor modulators in Peyronie’s disease models

Background: Peyronie's disease (PD) is a fibrotic disorder of the penile tunica albuginea, characterised by the formation of a localised fibrous plaque that can lead to deformity and erectile dysfunction. Nonsurgical therapeutic options for PD are limited in efficacy and safety. Myofibroblasts are key cells in the pathogenesis of PD, and inhibition of myofibroblast transformation has been suggested as a therapeutic option. Objective: To identify potential drugs using a novel phenotypic assay and then to test them using in vitro and in vivo models of PD. Design, setting, and participants: We have developed and validated a phenotypic screening assay that measures myofibroblast transformation, by which we tested 21 compounds that were suggested to be efficacious in treating PD. The successful hits from this assay were further tested using in vitro and in vivo models of PD. Results and limitations: The new assay was able to detect transforming growth factor-β1–induced myofibroblast transformation. Using this assay, phosphodiesterase type 5 inhibitors (PDE5i) and selective oestrogen receptor modulators (SERMs) were identified to significantly inhibit myofibroblast transformation. A PDE5i (vardenafil) and an SERM (tamoxifen) inhibited myofibroblast transformation, collagen gel contraction, and extracellular matrix production in a synergistic fashion. In a rat model of PD, the antifibrotic effect of the combination of vardenafil and tamoxifen was greater than that of each drug alone. This study is limited by not providing a molecular mechanism for the proposed synergy. Conclusions: This is the first demonstration of a synergistic activity between a PDE5i and an SERM discovered through a phenotypic screening approach. Future clinical trials using a combination of these drugs should be considered during the active phase of PD, given the early evidence of benefit in both in vitro and in vivo models. Patient summary: This report suggests that the combination of a phosphodiesterase type 5 inhibitor and a selective oestrogen receptor modulator may be efficacious in treating Peyronie's disease in its active phase

High magnification deconvolved images for WNT4, MMP7, CD1 and c-MYC co-expression in control and malignant penile tissue.

<p>Composite, representative, micrographs of immunofluorescently stained penile tissue cores used to quantify co-localization of four proteins WNT4 (Alexa 405-blue), MMP7 (FITC-green), CD1 (Cy3-orange) and c-MYC (Cy5-red) after deconvolution. Tissue cores from 8 patients (control), 25 malignant tissue cores (11 grade I and 14 grade II) were imaged using an Olympus FV1000 confocal microscope. High magnification imaging (at 1024x1024 resolution) was performed for deconvolution analysis using Huygens Profesional software. Areas of squamous cell carcinoma in malignant tissue cores were identified, visually (see box) from a serial H&E section of the tissue, and imaged at high magnification (40x with 4–6x zoom). Two representative immunofluorescently labelled cores are shown: A is control and B and C are grade I and grade II, squamous cell carcinoma samples, respectively; Inset is H&E serial sections of at low magnification. High magnification images were deconvolved to reduce noise and converted to 16bit tif files for each fluorophore (Alexa Fluor-405, FITC, Cy3 and Cy5) were imported into Huygens deconvolution software and pseudo colored (FITC, green; Alexa Fluor-405, blue; Cy3, orange; and Cy5, red). Z-projections for up to 53 images for each tissue core. Representative images of controls and malignant carcinoma samples are shown. Scale bar = 3μm. High magnification images were used for quantitative co-localization measurements.</p

Box plot of differential pattern for WNT4, MMP7, CD1 and c-MYC co-expression in control and malignant penile tissue.

<p>Quantitative co-localization was performed from high magnification images (e.g. as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#pone.0124395.g003" target="_blank">Fig 3</a>) and image files used for quantitation as described in materials and methods in WCIF ImageJ software co-localization plugin to measure the Pearson coefficient of co-localization. 35 individual tissue cores that included control (gradient from centre ) and malignant grade I (gradient from left to right ) and grade II (gradient from right to left ) were used for each fluorophore to calculate the co-localization of expression of WNT4, MMP7, CD1 and c-MYC, generating 12 comparisons: CD1 and WNT4 (orange/blue), MMP7 and WNT4 (green/blue), c-MYC and WNT4 (pink/blue), CD1 and c-MYC (orange/pink), MMP7 and c-MYC (green/pink), MMP7 and CD1 (green/orange). Significance of difference in the Pearson coefficient of co-localization between control and grades I and II (*0.02–0.04) and between grade I and grade II (§ p<0.04, range 0.04–0.0001) samples was calculated using the Mann-Whitney U test; boxes showing comparisons without symbols are not significantly different.</p

Representative images from Zeiss AxioScan Z1 slide scanner.

<p>Composite, overlay, of four fluorophores (FITC, Alexa Fluor-405, Cy3 and Cy5 for MMP7, Wnt4, CD1 and c-MYC, respectively) in (A) PeCa tissue cores (core B2 of PE241) and (B) control (core D5 of PE241, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124395#sec002" target="_blank">methods</a>). C-F are individual images used to construct the composite of PeCa tissue core, for MMP7 (C, green), Wnt4 (D, blue), CD1 (E, orange) and c-MYC (F, red). G-J are individual images used to construct the composite of control tissue core, for MMP7 (G, green), Wnt4 (H, blue), CD1 (I, orange) and c-MYC (J, red). Quantitative analysis was performed on the non-enhanced, original, gray images for which all settings were identical in all tissue core images analyzed. Scale bar = 250 μm.</p