Foreskin Specimens Research Articles

MP78-06 COLLAGEN IS MORE ABUNDANT AND STRUCTURALLY ALTERED IN LICHEN SCLEROSUS

You have accessJournal of UrologyCME1 Apr 2023MP78-06 COLLAGEN IS MORE ABUNDANT AND STRUCTURALLY ALTERED IN LICHEN SCLEROSUS Ethan Richmond, Melissa Champer, Eduardo Mora, Wei Huang, Paul Campagnola, and Matthew D Grimes Ethan RichmondEthan Richmond More articles by this author , Melissa ChamperMelissa Champer More articles by this author , Eduardo MoraEduardo Mora More articles by this author , Wei HuangWei Huang More articles by this author , Paul CampagnolaPaul Campagnola More articles by this author , and Matthew D GrimesMatthew D Grimes More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003355.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lichen sclerosus (LS) is a chronic inflammatory disease of the genital epithelium that results in fibrosis and severe urethral stricture disease in affected men. Altered physical properties of fibrotic tissue may result from increased collagen content (generally from increased production) and/or from altered collagen structure (generally from fiber cross-linking) and are undefined in LS. We aim to test the hypothesis that genital skin and male urethra affected by LS has increased collagen content and altered collagen structure. METHODS: Collagen was stained with picrosirius red in human urethral, vulvar, and foreskin specimens with and without LS. An observer blinded to LS status and tissue type acquired three 20x stromal images from each specimen. Using Image J software, we quantified and compared 1) collagen content (using two metrics: collagen proportionate area [CPA] and collagen fiber count), 2) collagen fiber length and width, and 3) collagen structure using the texture analysis technique gray level co-localization matrix (GLCM) with respect to LS status and tissue type. Pairwise comparisons were made using the Student’s T test with respect to LS status and tissue type. RESULTS: We analyzed 23 LS specimens (vulva n=9, urethra n=7, foreskin n=7) and 29 non-LS specimens (vulva n=9, urethra n=7, foreskin n=13). Fiber count and CPA were significantly higher in all LS specimens compared to non-LS specimens: CPA mean (SD): LS 0.971 (0.03) vs no-LS 0.948 (0.02), p<0.007; fiber count mean (SD): LS 2906 (127) vs no-LS 2509 (78) fibers; p=0.003 (Figure 1). Collagen fiber width and length were similar with respect to LS status. GLCM analysis showed decreased inverse difference moment and increased entropy in LS tissues indicative of less homogeneous and more disorganized tissue structure (p<0.001) (Figure 1). CONCLUSIONS: LS tissues have increased collagen content compared to non-LS tissues. Quantitative assessment of collagen organization, using GLCM, revealed less homogeneity and more disorganization of collagen in LS compared to non-LS tissues. Taken together, our findings suggest that alterations in physical tissue properties seen in LS may be due to both increased collagen abundance and altered structure. Source of Funding: K12DK100022NIH/NIDDK © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1133 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ethan Richmond More articles by this author Melissa Champer More articles by this author Eduardo Mora More articles by this author Wei Huang More articles by this author Paul Campagnola More articles by this author Matthew D Grimes More articles by this author Expand All Advertisement PDF downloadLoading ...

Collagen is More Abundant and Structurally Altered in Lichen Sclerosus

To test the hypothesis that genital skin and male urethra affected by lichen sclerosus (LS) has increased collagen content and altered collagen structure. We used picrosirius red to stain and image collagen in human urethral, vulvar, and foreskin specimens with and without LS. Using Image J software, we quantified and compared (1) collagen content (using 2o metrics: collagen proportionate area [CPA] and collagen fiber count), (2) collagen fiber length and width, and (3) collagen structure using the texture analysis technique gray level co-localization matrix (GLCM) with respect to LS status and tissue type. We analyzed 23 LS specimens (vulva n=9, urethra n=7, foreskin n=7) and 29 non-LS specimens (vulva n=9, urethra n=7, foreskin n=13). Fiber count and CPA were significantly higher in all LS specimens compared to non-LS specimens (CPA: mean±SD 0.971±0.03 vs 0.948±0.02, P < .007; fiber count: mean±SD=2906±127 vs 2509±78 fibers; P=.003). Collagen fiber width and length were similar with respect to LS status. GLCM analysis showed decreased inverse difference moment and increased entropy in LS tissues indicative of less homogeneous and more disorganized tissue structure (P<.001). LS tissues have greater collagen content compared to non-LS tissues. Quantitative assessment of collagen organization, using GLCM, revealed less homogeneity and more disorganization of collagen in LS compared to non-LS tissues. Taken together, our findings suggest that alterations in physical tissue properties seen in LS may be due to both increased collagen abundance and altered structure.

Histological Characteristics of Skin Treated With a Fractionated 1064-nm Nd: YAG Picosecond Laser With Holographic Optics.

Picosecond lasers (PSL) constitute a significant technological advancement and exert rejuvenating effects upon the skin. This study was conducted to investigate changes in the skin upon treatment with the fractionated 1064-nm Nd: YAG PSL through in vivo and ex vivo human histological analysis. In vivo back skin specimens were treated with a fractionated 1064-nm PSL at 1.3, 2.1, and 2.9 mJ fluence for two passes, and 2.9 mJ for 10 passes, and then stained with hematoxylin and eosin (H&E). Ex vivo foreskin specimens after circumcision surgery were treated with a PSL at 1.3, 2.1, and 2.9 mJ fluence for two and 10 passes, followed by H&E staining. Ex vivo skin tissue sections treated with a PSL at 2.9 mJ fluence for 10 passes were also immunostained for Melan-A and CD31. Intraepidermal vacuoles were observed, along with pigment accumulation and inflammatory cell infiltration in the vacuoles at 24 hours after PSL treatment in the in vivo skin specimens. The vacuoles expanded as the fluence increased. Numerous intraepidermal vacuoles were observed, with dermal hemorrhage and inflammatory cell infiltration upon high-fluence, multi-pass PSL treatment in the in vivo skin specimens. PSL treatment yielded both epidermal and dermal vacuoles in ex vivo skin specimens. Melan-A-positive cells were seen in the cystic wall of vacuoles in the epidermal basal layer, whereas CD31-positive cells were detected in the cystic wall of some dermal vacuoles. The fractionated 1064-nm PSL produced epidermal vacuoles and dermal lesions, with histological differences between the in vivo and ex vivo skin specimens. Lasers Surg. Med. © 2021 Wiley Periodicals LLC.

Application of reflectance confocal microscopy to investigate the non-ablative, micro-ablative, and ablative effects of CO2 fractional laser irradiation on skin.

CO2 fractional laser, as an ablative fractional laser, is commonly used in cosmetic treatment. We applied CO2 fractional laser irradiation to skin in vitro and used reflectance confocal microscopy (RCM) to image and detect the presence of any non-ablative, micro-ablative and ablative effects, in order to better understand the features of a CO2 fractional laser. In vitro irradiation of foreskin was performed using a CO2 fractional laser. Foreskin specimens were divided into 4 groups that received different amounts of irradiation energy, based on the number of irradiation passes they received: 5, 10, 15, and 20 passes, respectively. This corresponds to fluence energy of 16.3, 32.6, 48.9, 65.3 J/cm2. Immediately after irradiation, digital microscopy (DM), RCM, and histopathology were performed to observe whether the non-ablative, micro-ablative, and ablative phenomenon occurred, and the injury features of MTZs. Immediately after CO2 fractional laser irradiation, RCM and DM showed that when the numbers of passes were 5 and 10, a micro-ablative column (MAC) could not be observed or was very small. We mainly observed a thicker thermal coagulation zone (TCZ), representing non-ablative or micro-ablative effects. When the number of passes were increased to 15 and 20, the MAC was significantly enlarged and surrounded by a TCZ of medium thickness, representing ablative effects. For the first time, this study used RCM and DM to demonstrate that a CO2 fractional laser could achieve non-ablative, micro-ablative, and ablative effects on irradiated skin via different energy levels.

Androgen Receptor Expression in Hypospadias.

Introduction:The exact mechanism behind the development of hypospadias is unclear. Research studies on androgen receptor (AR) expression are controversial with results stating all possible outcomes – AR elevated, similar, or reduced when compared to normal.Aims:The aim is to study the AR expression and hormone levels in hypospadias patients and compare them with children having normal genitalia.Methods:Group 1 (controls) involved patients who underwent circumcision for phimosis while Group 2 involved hypospadias patients who did not receive any preoperative testosterone. Preoperative hormonal assay included luteinizing hormone, follicle-stimulating hormone, and free testosterone levels in all the patients. The foreskin specimen was analyzed for AR expression using immunohistochemistry (anti-AR antibody PathnSitu, clone R441, 1/100 dilution). AR staining was expressed as H score. The H score was calculated by multiplying the intensity of staining and the percentage of stained cells showing cytoplasmic positivity at high power (×40).Results:There were 27 patients in Group 1 while 16 in Group 2 (distal 10; proximal 6).There was no significant difference in the age distribution. The mean H score was significantly higher (189.5) in hypospadias patients compared to controls (97.5) and was significantly higher in proximal (220) compared to distal (159) hypospadias. There was no significant difference in hormone levels between groups.Conclusion:AR expression was significantly elevated in hypospadias patients. It was higher in proximal compared to distal hypospadias, probably due to end-organ overexpression. Further larger trials are likely to through light into this controversial subject.

Nonlinear viscoelastic properties of native male human skin and in vitro 3D reconstructed skin models under LAOS stress

This work discusses the first set of rheometric measurements carried out on commercially accessible juvenile and aged skin models under large amplitude oscillatory shear deformations. The results were compared with those of native male whole human and dermis-only foreskin specimens, catering to a few ages from 0.5 to 68 years, including specimens from a 23-year-old male abdomen. At large strains, strain thinning was more pronounced for the dermis of the young skins and for their whole skin counterparts. An inverse qualitative tendency was observed for the adult skins and the skin models. This can be explained by the high dermal collagen compactness associated with an incomplete epidermal proliferation. The qualitative Lissajous plots as well as the quantitative dimensionless indices analyzed using the MITlaos software indicated predominant nonlinear intracycle elastic strain stiffening and viscous shear thinning for all the native specimens at the maximum deformation. For the full thickness models, we have evidence of structure collapse and yielding under similar conditions. The whole skin specimen from the 68-year-old male showed smaller age-dependent nonlinear elastic contributions than the dermis, which we relate to the epidermal degeneration taking place during aging. Regardless of the age group, the models manifested more pronounced intercycle and intracycle elastic nonlinearities, and their magnitudes were significantly larger. The nonlinear elastic trends will serve as advanced standards for understanding and delineating the mechanical limits of destructive and non-destructive deformations of such unique biomaterials.

Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105+ fibroblast cells through the Smad and MAPK pathways.

The skin, as the largest organ of the human body, is an important source of stromal stem cells with multipotent differentiation potential. CD105+ mesenchymal stem cells exhibit a higher level of stemness than CD105− cells. In the present study, human dermal-derived CD105+ fibroblast cells (CD105+ hDDFCs) were isolated from human foreskin specimens using immunomagnetic isolation methods to examine the role of bone morphogenetic protein (BMP)-7 in osteogenic differentiation. Adenovirus-mediated recombinant BMP7 expression enhanced osteogenesis-associated gene expression, calcium deposition, and alkaline phosphatase activity. Investigation of the underlying mechanisms showed that BMP7 activated small mothers against decapentaplegic (Smad) and p38/mitogen-activated protein kinase signaling in CD105+ hDDFCs. The small interfering RNA-mediated knockdown of Smad4 or inhibition of p38 attenuated the BMP7-induced enhancement of osteogenic differentiation. In an in vivo ectopic bone formation model, the adenovirus-mediated overexpression of BMP7 enhanced bone formation from CD105+ hDDFCs. Taken together, these data indicated that adenoviral BMP7 gene transfer in CD105+ hDDFCs may be developed as an effective tool for bone tissue engineering.

CCR5 HIV-1 receptor predominance in adult male prepuce; the major entry route for HIV-1 in indigenous black males in zambia?

HIV-1 entry requires not only CD4 molecule but also CCR5 (CD 195) and CXCR4 (CD 184) coreceptors. A number of randomised controlled trials in Africa have reported that male circumcision (MC) reduces the risk of HIV-1 acquisition by up to 60%. Other studies have reported that sexually transmitted infections (STIs) increase the risk of infection by HIV via an inflammatory recruitment of more HIV target cells to the foreskin. Our aim was to compare the density of HIV-1 co-receptors (CCR5 and CXCR4) in naïve penile prepuce of neonates and penile prepuce of adults with and without history of ulcerative STIs at Male Circumcision Centres in Lusaka, Zambia. Twenty (20) fresh foreskin samples were included: five (5) from neonates, ten (10) from adult males without history of ulcerative STIs and five (5) from adult males with a history of ulcerative STIs. Immediately following MC, fresh foreskin specimens were fixed using 10% normal buffered formalin and transported to University Teaching Hospital (UTH) where tissues were processed and stained with anti-CD 195 and anti-CD 184 antibodies. Neonatal penile foreskin co-receptor mean density for CCR5 and CXCR4 was 13±5.148/mm2 and 7±1.581/mm2 respectively. CCR5 mean density of adults without past history of ulcerative STIs was 42.1±11.874/mm2 while those with history of ulcerative STIs was 78.6±13.520/mm2. Densities of CCR5 were all statistically significant with both having Pvalue of 0.000. CXCR4 mean density was 18.6±4.812/mm2 in adults without past history ofulcerative STIs and 23.4±4.393/mm2 in those with history of ulcerative STIs giving an insignificant P-value of 0.084. It could be concluded that CCR5 co-receptors provide major entry route for HIV-1 in male adults and that CCR5 seemed to be mobilized more than CXCR4 to the prepuce during inflammation. This supports evidence that MC reduces CCR5 co-receptors for acquisition and transmission of R5 strains of HIV-1.Key words: HIV, CCR5, CD 195, CD 184.

Effect of Magnesium Sulfate on Mitochondrial Oxygen Consumption Rate In vitro Study

Hypoxic insults initiate a cascade of biochemical events that result in irreversible neuronal damage. Magnesium sulfate agent has a possible neuroprotective effect as it can work at different stages of hypoxic brain injury. Objectives: Magnesium sulfate is used in the neonatal management of HIE as an adjunct medication to reduce Hypoxic Neuronal injury. In this Vitro study, we aimed to examine the efficacy of using magnesium sulfate in human cells to reduce oxygen consumption. We used in-vitro method utilizing foreskin as human cell surrogate. Method: Foreskin specimens from healthy newborns were collected immediately after circumcision and processed within 1 h for measuring the cellular rate of O2 consumption. Samples were placed in the oxygen phosphorescence analyzer and allowed to run for approximately 1 h. Injection then added magnesium sulfate at different doses into the vial. We analyze the O2 consumption in the samples at different levels of Magnesium Sulfate; the results were plotted using Kaleida Graph TM software. Results: The rate of respiration reduced with increasing the dose of MgSo4. The Cumulative analysis of cellular respiration rate was before and after an addition of MgSO4. Collectively yielding a (k) value of 0.08 μM O2/min μM O2 min-1 mg- and 0.04 M O2/min μM O2 min-1 mg- respectively with a significant P-value of <0.001. Conclusion: Magnesium sulfate reduces the rate of O2 Consumption in a dose-dependent manner.

Does Routine Pathology Analysis of Adult Circumcision Tissue Identify Penile Cancer?

To assess the utility of foreskin pathology analysis, we evaluated the outcomes and the costs of this practice in patients for whom penile cancer was not suspected. Adult circumcision specimens are routinely sent for pathologic analysis even when penile cancer is not suspected, increasing costs with little benefit. All adult patients who underwent circumcision between January 2000 and August 2013 at a single institution were evaluated by retrospective chart review. Cases of suspected penile cancer (n = 6) were excluded. We identified cases where foreskin specimens were sent for pathologic analysis and reviewed pathology reports. Our Department of Pathology estimated the cost for evaluation of specimens at $311 per case. A total of 147 circumcisions were performed in patients with no suspicious findings. Pathologic analysis was obtained in 69% (101 of 147) of the cases. Inflammation (58%) was the most common finding. One unsuspected instance of squamous cell carcinoma (Tis) was identified in a patient with human immunodeficiency virus (1 of 147 = 0.7%). The overall cost of pathologic analysis in this study was $31,411. In individuals without predisposing immunodeficiency and where cancer was not suspected, we found that pathologic analysis of circumcision specimens identified no additional malignancies. Our data suggest that in this normal risk population, pathologic analysis may not be required. Additionally, forgoing pathology on foreskin specimens in lower risk cases may reduce costs to the health care system.

The Value of Pathological Examination of the Foreskin Following Circumcision

IntroductionWe determined the clinical impact and value of routine histopathological examination of the foreskin following circumcision. MethodsWe performed a retrospective study of 225 consecutive adult circumcisions. Indications for circumcision were categorized as benign or malignant based on preoperative clinical evaluation. Histopathological results were similarly classified as benign or malignant. Preoperative clinical impression and postoperative histological diagnosis were compared and reported as concordant (in agreement) or discordant (in disagreement). The cost impact of histopathology examination was analyzed with respect to study findings. ResultsOf the 225 patients 209 (92.9%) had clinically benign disease on preoperative evaluation and 16 (7.1%) had foreskin lesions suspicious for malignancy. Mean age was 57.0 years (range 23 to 92). Patients were younger in the benign group than in the malignant group (56.5 vs 62.8 years, p = 0.018). Black patients represented 65.8% of the study population and were similarly distributed between the 2 groups (p = 0.405). There was no statistical difference in patient height, weight, body mass index or comorbidities between the 2 groups. Preoperative clinical impression and postoperative histological diagnosis were concordant in all 209 patients in the benign group. Of the 16 patients suspected to have malignant disease preoperatively 9 (56.2%) had malignancy and 7 (43.8%) had benign disease on histopathological examination. ConclusionsRoutine histological examination of a foreskin specimen in the absence of clinical suspicion for malignancy appears to have diminished benefit in the setting of benign preoperative indications. Omitting this traditional practice in patients with benign surgical indications may positively impact health care costs without compromising quality of care.

Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast Rich Culture = إستهلاك الأوكسجين في المايتوكوندريا من قبل القلفة و الخلايا الليفية

This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures. Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 μM O2 min(-1) mg(-1) (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 μM O2 min(-1) per 10(7) cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation.

Quantitative measurement of the androgen receptor in prepuces of boys with and without hypospadias

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Despite diverse anatomical and histological trials in humans and animal models, the aetiology of hypospadias remains unknown and currently there is no clear molecular explanation about the emergence of this disease; however, genetic, endocrine and environmental mechanisms have been suggested. The aim of the present study was to quantify and compare the androgen receptor (AR; mRNA and protein) levels in 40 prepuces of boys with and without hypospadias using quantitative real-time polymerase chain reaction, Western Blot and standardised, automated immunohistochemistry. AR mRNA (P = 0.013) and AR protein (P = 0.014) was significantly elevated in the prepuces of boys with hypospadias compared with boys without hypospadias. Altogether our data indicate that elevated AR mRNA and protein levels can be considered as a biochemical response of an AR signalling defect as an identified cause in boys with hypospadias. Additionally, nuclear staining intensity for AR-protein in specimens of boys with hypospadias was higher than in boys with phimosis. To address the role of the androgen receptor (AR) on mRNA and protein levels in prepuces of boys with and without hypospadias. Data from 40 foreskin specimens of consecutive circumcised boys (20 with vs 20 without hypospadias) were enrolled in this prospective study. After surgery, samples were fixed in formaldehyde and frozen in liquid nitrogen. Total RNA was isolated from frozen tissue and transcribed to complementary DNA. The amount of AR mRNA was measured by quantitative real-time polymerase chain reaction and Western Blot and standardised, automated immunohistochemistry were used to assess AR protein levels. The mean age at time of surgery was 61.8 and 30.9 months in boys without and with hypospadias, respectively. There was penile, coronal and sine hypospadias in seven (35%), nine (45%), and four (20%) boys, respectively. AR mRNA was significantly elevated in the prepuces of boys with hypospadias compared with boys without hypospadias, at a mean (sd) of 28.33 (5.39) vs 15.31 (1.85) (P = 0.013). Furthermore, the amount of AR protein was higher in boys with, compared with boys without hypospadias, at a mean (sd) of 133.25 (6.17) vs 100 (4.45) (P = 0.014). Different AR mRNA expression and protein levels seem to be an indication of an AR signalling defect as a cause in boys with hypospadias. Decreased AR DNA binding and functional capability may result in a compensatory up-regulation of both AR mRNA and protein. Further studies are necessary to perform structural analysis of the AR and to corroborate these preliminary findings.

High-risk Human Papilloma Virus Infection of the Foreskin in Asymptomatic Boys

To evaluate the prevalence and genotypes of high-risk human papilloma virus (HPV) infection of the foreskin in asymptomatic boys before first sexual intercourse. We collected 50 consecutive foreskin specimens after radical circumcision. Indication for surgery was phimosis. High-risk HPV status was determined by real-time polymerase chain reaction for the genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. Immunohistochemistry for p16(INK4a) was performed. The median age at the time of surgery was 5.5 years (range, 5 months-15 years). High-risk HPV was detected in 6 of 50 foreskins (12%). All positive samples showed HPV16. No association with age or grade of phimosis was observed. Two samples were focally positive for p16(INK4a), both of which were HPV-negative. In a significant proportion of boys, subclinical high-risk HPV infections are found in the foreskin, which could be a reservoir for HPV-associated diseases. Our study generates the hypothesis that nonsexual routes play significant roles in HPV transmission. Because the human foreskin represents a high-risk HPV reservoir, vaccination may be also advised in boys.

Comparison of Gene Expression Profiles between Keratinocytes, Melanocytes and Fibroblasts

BackgroundThe skin has many important functions such as protection, preservation, temperature regulation, and vitamin D synthesis. It is composed of a variety of cell types including keratinocytes, melanocytes and fibroblasts.ObjectiveWe attempted to compare the gene expression profiles between keratinocytes, melanocytes and fibroblast, using cDNA microarray.MethodsKeratinocytes, melanocytes and fibroblasts were primary cultured from five foreskin specimens. Total RNAs were extracted and pooled to reduce the individual variations, and then used for cDNA microarray.ResultsTotal 12,028 genes were selected as the reliable genes whose expression was detected in at least one of the three cell types. By comparing the relative expression levels with cutoff limitation as a fourfold change, we obtained 126 fibroblast-specific, 179 keratinocyte-specific and 173 melanocyte-specific genes, many of which are known to be characteristically expressed in each cell type. In addition, we identified many genes whose skin-specific functions have not yet been determined.ConclusionOur data provide important information on which to base further investigation into the specification of skin cell types.

Insertion/Deletion Polymorphisms in the ΔNp63 Promoter Are a Risk Factor for Bladder Exstrophy Epispadias Complex

Bladder exstrophy epispadias complex (BEEC) is a severe congenital anomaly; however, the genetic and molecular mechanisms underlying the formation of BEEC remain unclear. TP63, a member of TP53 tumor suppressor gene family, is expressed in bladder urothelium and skin over the external genitalia during mammalian development. It plays a role in bladder development. We have previously shown that p63−/− mouse embryos developed a bladder exstrophy phenotype identical to human BEEC. We hypothesised that TP63 is involved in human BEEC pathogenesis. RNA was extracted from BEEC foreskin specimens and, as in mice, ΔNp63 was the predominant p63 isoform. ΔNp63 expression in the foreskin and bladder epithelium of BEEC patients was reduced. DNA was sequenced from 163 BEEC patients and 285 ethnicity-matched controls. No exon mutations were detected. Sequencing of the ΔNp63 promoter showed 7 single nucleotide polymorphisms and 4 insertion/deletion (indel) polymorphisms. Indel polymorphisms were associated with an increased risk of BEEC. Significantly the sites of indel polymorphisms differed between Caucasian and non-Caucasian populations. A 12-base-pair deletion was associated with an increased risk with only Caucasian patients (p = 0.0052 Odds Ratio (OR) = 18.33), whereas a 4-base-pair insertion was only associated with non-Caucasian patients (p = 0.0259 OR = 4.583). We found a consistent and statistically significant reduction in transcriptional efficiencies of the promoter sequences containing indel polymorphisms in luciferase assays. These findings suggest that indel polymorphisms of the ΔNp63 promoter lead to a reduction in p63 expression, which could lead to BEEC.

Dermatopathology of the foreskin: an institutional experience of over 400 cases

Diseases of the foreskin may manifest with an array of pathologic findings, including potentially under-recognized dermatologic conditions. Herein, we summarize an institutional experience in foreskin dermatopathology. Diagnoses rendered on foreskin specimens between 1982 and April 2009 were obtained through a computer-based keyword search. Cases given normal, non-specific or descriptive diagnoses were reviewed by a dermatopathologist. Keyword search yielded 414 foreskin diagnoses. Interpretations included normal foreskin (n = 131), benign lesions (n = 262) and malignant/dysplastic entities (n = 21). Of 353 cases given normal, descriptive or non-specific diagnoses, 334 were reviewed. Of reviewed cases, 209 (63%) were given more specific diagnoses [e.g. spongiotic dermatitis (n = 115), lichen sclerosus et atrophicus (LSA; n = 41), interface/lichenoid dermatitis (n = 26), psoriasiform dermatitis (n = 7)]. Discrepancy between the clinical and pathologic impression was frequently noted (n = 77). This study shows benign inflammatory lesions represent the most frequent foreskin pathology. When possible, specific diagnoses should be rendered, as accurate classification may be of clinical importance. There is an abundance of recent literature on the role of circumcision in disease prevention, and this topic is explored. We discuss the theoretical possibility that foreskin inflammation compromises the mucosal/epithelial barrier, thus playing a role in disease transmission.

Basaloid squamous cell carcinoma presenting as paraphimosis of the foreskin: a case report

A 63-year-old male presented with a pharaphimosis, a constricting ring and a firm depressed ulcer of his foreskin. The histopathology of the foreskin specimen revealed a basaloid squamous cell carcinoma (BC) arising in the mucosa with peri-neural and lymphovascular invasion. The tumour cells were p16, p63, 34BE12, CD34 and CD31 positive. Ultrastructurally there were a few intracytoplasmic tono-filaments and desmosomes, however neurosecretory dense core granules were absent. Three years earlier the patient underwent a total laryngectomy and neck dissection for a large supraglottic moderately differentiated squamous cell carcinoma (T3N2B). Basaloid carcinoma is a recognised unusual variant of squamous cell carcinoma of the penis. It usually occurs in the glans, and is frequently associated with human papilloma virus. To our knowledge there have been no reported cases of BC arising in the foreskin. Given the unusual location of this BC, the discussion as to whether this foreskin lesion represents a metachronous primary BC or an unfortunate metastasis of the laryngeal SCC arises. We favour this lesion to be a primary BC of the foreskin.

Suspected penile cancer: a method to improve handling of pathology specimens

BACKGROUND A major problem encountered by the supranetwork team managing new cases of penile cancer is inadequate tissue preparation for pathological evaluation of specimens. The histological examination of a circumcision or wide local excision specimen, when there is a clinical suspicion of malignancy, relies on receiving an orientated and pinned-out specimen [1]. When a foreskin is placed directly into formalin the specimen immediately contracts. After fixation it is not possible to stretch the specimen to identify the dorsal slit, or to take representative sections for a histological assessment of surgical margins. Similarly, excision-biopsy specimens might be difficult to orientate. We describe a method that enables a foreskin and excision-biopsy specimens to be pinned and orientated using material readily available in any operating theatre. METHODS In combination with the pathologist, we have developed a method that maintains the integrity of the specimen and allows orientation (Figs 1 and 2). The pathologist can then identify the dorsal slit, which is not a true surgical margin. The distal (glans) and proximal (shaft) margins can be identified and marked by using two colours of ink. The specimen is sliced sagittaly and the location of each sample identified using a block code. This block code must appear in the text of the pathology report, as these specimens are often reviewed in a distant cancer centre which will not have access to any block code written on the form at the time of sectioning. DISCUSSION About 80% of penile carcinomas arise from the foreskin and/or glans [2]. The aim of surgical management is to achieve control of disease without compromising functional or cosmetic results; hence organ-sparing procedures are increasingly favoured. Traditionally, a surgical margin of 20 mm has been advised. However, one study examined men who had conservative treatment for carcinoma of the penis [3]; the surgical resection margin was <10 mm in 48% of patients and <20 mm in 90% of patients. After a median follow-up of 26 months, 4% were found to have recurrence. This suggests that surgical margins within a few millimetres of microscopic tumour are sufficient. To accurately identify surgical margins, a clear orientation of the specimen is vital; the above technique describes a simple and cost-effective method.

Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.