P63 Expression Research Articles

Abstract PO-040: Comparing digital image analysis with a manual scoring approach for quantification of p63 and BRCA1 protein expression in oropharyngeal squamous cell carcinoma

Abstract In oropharyngeal squamous cell carcinomas (OPSCC) there is overexpression of p63. We previously identified a link between p63 and BRCA1 in OPSCC, both at a gene and protein level. However, interpretation of biomarkers based on protein expression identified using immunohistochemistry can be subject to interobserver variability. We aimed to explore the option of digital image analysis as an alternative approach to immunohistochemical protein quantification. Herein, we compare manual histological interpretation of p63 and BRCA1 expression in the context of HPV status with digital image analysis. Representative samples of a clinically annotated cohort of OPSCC (n=191) were arranged in triplicate tissue microarrays (TMA) and stained immunohistochemically for p63 (p63NCL and p63DN) and BRCA1 (D-9 and Ab1). RNA in situ hybridization for a cocktail of 18 high-risk HPV genotypes (HR-18 HPV) was used to determine HPV status. Cases were scored manually using at least two independent scorers to generate consensus Q scores. Each TMA slide was digitally scanned. These images were imported into QuPath version 0.2.3, which was used to quantify the expression of each biomarker and generate digital H scores. Statistical analysis was conducted using R version 4.2.3 and GraphPad Prism5. Spearman’s Rank correlation was used to compare the digital and manual scores for each biomarker. Kaplan-Meier curves and log rank tests were used for survival analysis. Preliminary analysis showed a strong correlation between manual scoring methods and digital scores for each of the four biomarkers: p63NCL (rs=0.77, p<0.0001); p63DN (rs=0.72, p<0.0001); BRCA D9 (rs=0.75, p<0.0001) and BRCA AB1 (rs=0.84, p<0.0001). We identified a subgroup of HPV negative patients with low p63NCL expression and high BRCA D9 expression that had poor 5-year overall survival (p<0.0001). The process of digital quantification is more labour intensive than one might anticipate. It requires significant pre-processing to remove artefacts prior to obtaining a computer-generated H score. In contrast manual Q scoring allows immediate assessment. In addition, the BRCA D-9 antibody had non-specific staining, highlighting darkly staining “dendritic-like” cells which complicated digital assessment. The results show the effectiveness and reproducibility of digital pathology scoring methods for antibody quantification when compared with traditional manual scoring methods. However manual scoring methods presently used by pathologists are significantly more time efficient when compared to newer digital techniques. There is a need for improved or automated pre-processing techniques to remove some of the pitfalls associated with this approach for future studies validating use of combined low p63NCL expression and high BRCA D9 expression to predict outcome in patients with OPSCC. Citation Format: Laura Graham, Stephanie Craig, Kris McCombe, Stephen McQuaid, Simon McDade, Jacqueline James. Comparing digital image analysis with a manual scoring approach for quantification of p63 and BRCA1 protein expression in oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-040.

Effects of bisphenol A on murine salivary glands and human tumor cell lines

Bisphenol A (BPA) is an endocrine-disrupting chemical with a potential role in endocrine cancers. However, the effects of BPA on the salivary glands have been barely explored. We investigated the impact of in vivo sub-chronic exposure to BPA and its in vitro effects on human salivary gland mucoepidermoid carcinoma cell lines. Male and female mice were exposed to BPA (30 mg/kg/day). Sublingual and submandibular salivary glands from an estrogen-deficiency model were also analyzed. BPA concentration in salivary glands was evaluated by gas chromatography coupled to ion trap mass spectrometry. Immunohistochemical analysis using anti-p63 and anti-α-SMA antibodies was performed on mouse salivary gland tissues. Gene expression of estrogen receptors alpha and beta, P63 and α-SMA was quantified in mouse salivary gland and/or mucoepidermoid (UM-HMC-1 and UM-HMC-3A) cell lines. Cell viability, p63 and Ki-67 immunostaining were evaluated in vitro. BPA disrupted the tissue architecture of the submandibular and sublingual glands, particularly in female mice, and increased the expression of estrogen receptors and p63, effects that were accompanied by significant BPA accumulation in these tissues. Conversely, ovariectomy slightly impacted BPA-induced morphological changes. In vitro, BPA did not affect the proliferation of neoplastic cells, but augmented the expression of p63 and estrogen receptors. The present data highlight a potential harmful effect of BPA on salivary gland tissues, particularly in female mice, and salivary gland tumor cells. Our findings suggest that estrogen-dependent pathways may orchestrate the effects of BPA in salivary glands.

P63 immunoexpression in hair follicles of normal and alopecia X-affected skin of Pomeranian dogs.

Alopecia X in Pomeranians is caused by a hair cycle deregulation, associated with downregulation of key regulatory genes of the Wnt and Shh pathways, and stem-cell markers. However, the pathogenesis remains unclear. p63 is an important transcription factor correlated with the aforementioned hair cycle modulating genes. The aim of this study was to highlight possible changes of p63 immunohistochemical expression within the hair follicles in canine alopecia X compared with normal skin. Skin biopsies from 19 alopecia X-affected and six control Pomeranians were analysed. Serial histological sections of skin biopsies harbouring anagen, telogen and kenogen hair follicles were immunohistochemically evaluated for differences in p63 expression in the affected and control samples. Dogs with alopecia X had a significantly decreased immunoexpression of p63 in telogen and kenogen hair follicles. The decrease of p63 immunoexpression observed in canine alopecia X suggests an involvement of p63 in hair cycle.

P63 and Ki-67 expression in radicular cyst

ObjectivesThe aim of the current study was to identify the expression of P63 and its relation to odontogenic epithelial cell proliferation, severity of the inflammatory infiltrate and size of radicular cysts (RCs). MethodsIn this retrospective cross-sectional study, 30 cases of paraffin-embedded RCs were randomly selected from the archive. P63 and Ki-67 expression was assessed by immunohistochemistry. ResultsEpithelial P63 expression was absent in four (13.3%), weak in 10 (33.3%), and moderate in 16 (53.3%) cases. In the connective tissue wall of RC, P63 expression was absent in two (6.7%) cases, weak in 24 (80.0%) cases, and moderate in four (13.3%) cases. Ki-67 was found to be weakly expressed in 12 (40.0%) cases, moderately expressed in 13 (43.3%), and strongly expressed in five (16.7%) cases. No correlation was found between Ki-67 expression in odontogenic epithelium and P63 expression in the odontogenic epithelium (rho = 0.110, p = .563) or fibrous capsule (rho = 0.160, p = .399). Nevertheless, we found a positive correlation between Ki-67 expression in the odontogenic epithelium and the size of the RC (rho = 0.450, p = .013). The inflammatory infiltrate was negatively correlated with P63 expression in the odontogenic epithelium (rho = −0.428, p = .018), and with the size of cysts (rho = −0.728, p < .001). ConclusionsThere is a high expression of P63 throughout the odontogenic epithelium and connective tissue capsule of the RC. P63 expression in the odontogenic epithelium is negatively correlated with the degree of the inflammatory infiltrate but not with epithelial cell proliferation or the size of the cyst.

Study of Expression of p63 and Ki-67 in Normal, Dysplastic, and Malignant Lesions of Oral Mucosa

Background: The oral cavity is the most common site of cancer in the Indian subcontinent contributing to one-third of all cancers. The prognosis of oral dysplasia and malignant lesions can be predicted by both clinicopathological parameters and molecular markers. Aims and Objectives: The present study was undertaken to examine and compare the expression profile of cell cycle regulatory proteins p63 and Ki-67 in normal dysplastic and malignant lesions of oral mucosa and its correlation with prognosis. Settings and Design: This was a retrospective case–control study in a tertiary referral center. Materials and Methods: We evaluated and compared the expression profile of antigens p63 and Ki-67 on 60 cases of histologically proven oral dysplastic and malignant lesions oral squamous cell carcinoma (OSCC – 35 cases), oral epithelial dysplasia (OED – 15 cases), and 10 cases of benign and inflammatory oral lesions. Statistical Analysis: Mean and standard deviation were calculated and ANOVA statistical test was used to compare the data. Results: The mean labeling index of p63 and KI-67 was higher in OSCC than OED and minimum in inflammatory (controls) groups. Conclusion: p63 expression had a positive correlation with Ki-67 expression. Thus, p63 and Ki-67 may be a marker for tumorigenesis and tumor cell proliferation.

Stepwise activation of p63 and the MEK/ERK pathway induces the expression of ARL4C to promote oral squamous cell carcinoma cell proliferation.

Carcinogenesis is a multistep process wherein cells accumulate multiple genetic alterations and progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through a preneoplastic lesion/benign tumor to cancer. Histologically, oral squamous cell carcinoma (OSCC) progresses in multiple ordered steps that begin with mucosal epithelial cell hyperplasia, which is followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore hypothesized that genetic alteration-mediated multistep carcinogenesis would be involved in the development of OSCC; however, the detailed molecular mechanisms are unknown. We clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data from a pathological specimen of OSCC (including a non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion). The expression of numerous genes and signal activation were altered in the development of OSCC. Among these, the p63 expression was increased and the MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion and in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or the MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions, than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were frequently merged in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and MEK/ERK-MAPK cooperatively induce the expression of ARL4C and cell growth in OSCC cells. These results suggest that the stepwise activation of p63 and MEK/ERK-MAPK contributes to OSCC tumor cell growth through regulation of ARL4C expression.

Partial cellular reprogramming stably restores the stemness of senescent epidermal stem cells.

Adult stem cell senescence and exhaustion are important drivers of organismal age. Restored stem cell self-renewal has revealed novel therapeutic targets for decreasing the incidence of age-associated diseases (AADs) and prolonging the human health span. Transient ectopic expression of the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc (collectively known as OSKM) in somatic cells can induce partial cellular reprogramming and effectively ameliorate their age-associated hallmarks. However, how this form of rejuvenation is applied to senescent stem cells remains unknown. The Integrin-α6highCD71high epidermal stem cells (ESCs) with low self-renewal ability were sorted by flow cytometry and then treated by the interrupted reprogramming induced by transient expression of OSKM. The ability of secondary clones' generation and self-proliferation in vitro, as well as stem cell marker p63, were detected to determine their self-renewal ability. Besides, gene and protein of epidermal cell markers were detected to determine whether their cell identities were retained. Finally, DNA methylation age (eAge) and DNA dehydroxymethylase/methyltransferase were analyzed to explore the alternation of their global DNA methylation pattern during this rejuvenation. The partial reprogramming restored the youthful self-renewal and proliferation in senescent ESCs, including larger secondary clone generation, higher expression of stem cell marker p63 and proliferation marker Ki67, and faster proliferation speed, in each case without abolishing epithelial cellular identity. Moreover, the rejuvenation of adult stem cells could be maintained for 2 weeks after reprogramming factor withdrawal, which was more stable than that of differentiated somatic cells. Additionally, we found that partial reprogramming counteracted the acceleration of eAge in senescent epidermal stem cells and DNA methyltransferase 1 (DNMT1) may play a crucial role in this process. Partial reprogramming has high therapeutic potential for reversing adult stem cell age, providing an advanced way to treat AADs.

DOG-1 as a Myoepithelial Marker in Discriminating Invasive Breast Carcinomas From Non-Invasive Breast Lesions.

Introduction The most common cancer in women is, by far, breast cancer. The incidence and mortality of breast cancer must be reduced by a multidisciplinary strategy that includes education campaigns, preventive measures, screening programmes for early diagnosis, and the availability of treatment facilities. The use of immunohistochemical (IHC) stains with relative specificity for myoepithelial markers has become a mainstay of standard diagnostic breast pathology because the presence and distribution of myoepithelial cells might differ greatly amongst the distinct breast proliferation. Although it has also been reported that DOG1 is expressed in other mesenchymal tumours, DOG1 has been demonstrated to be sensitive and specific for the detection of gastrointestinal stromal tumors (GISTs). Both myoepithelial cells (MECs) and luminal epithelial cells have occasionally displayed DOG1 immunoreactivity in the breast. Materials and methods This prospective cross-sectional study was done in the Department of Pathology at Osmania General Hospital, Hyderabad on 60 cases from June 2017 to June 2019. Female patients with different breast lesions including benign proliferating lesions, ductal carcinoma in-situ (DCIS), and invasive carcinoma breast cases were included in the study.Inflammatory lesions, mesenchymal, and metastatic tumors were excluded.IHC expression of DOG1 as a myoepithelial marker to discriminate invasive from non-invasive breast lesions was evaluated and correlated withclinicopathological features. Results The mean age of the study population was 33.67 ± 8.48 in the benign group and 54.43 ± 12.84 in the malignant group. Fifty percent (15) of the patients with benign lesions belonged to the age group 20-30 years, whereas 26.7% (8) of the patients with malignant lesions belonged to the age group 61-70 years. DOG-1 expression was strongly positive in fibroadenoma, ductal hyperplasia, fibrocystic disease, whereas strongly negative in malignant disease of the breast (p< 0.0001). P63 expression was strongly positive in benign breast diseases and strongly negative in malignant diseases (p< 0.0001). Conclusion DOG1 seems to be similar to p63 as a myoepithelial cell marker both in normal breast tissue and in benign lesions. DOG1 is strongly positive in benign breast diseases and strongly negative in malignant breast diseases. Hence, it can be consideredas a useful myoepithelial marker in differentiating invasive breast carcinoma and non-invasive breast lesions.

Mineralocorticoid Receptor Mediates Cortisol Regulation of Ionocyte Development in Tilapia (Oreochromis mossambicus)

Cortisol is the predominant corticosteroid in ray-finned fish since it does not possess the aldosterone synthase necessary to produce specific mineralocorticoids. Cortisol is traditionally believed to function as a fish mineralocorticoid. However, the effects of cortisol are mediated through corticosteroid receptors in other vertebrates, and there is an ongoing debate about whether cortisol acts through the glucocorticoid receptor (GR) or the mineralocorticoid receptor (MR) in teleosts. To investigate this issue, we conducted a study using euryhaline Mozambique tilapia (Oreochromis mossambicus) as the experimental species. The experiment was designed to investigate the effect of cortisol on ionocyte development at both the cellular and gene expression levels in tilapia. We administered exogenous cortisol and receptor antagonists, used immunohistochemistry to quantify ionocyte numbers, and performed real-time PCR to assess the expression of the differentiation factor tumor protein 63 (P63) mRNA, an epidermal stem cell marker. We observed that cortisol increased the number of Na+-K+-ATPase (NKA)-immunoactive ionocytes (increased by 1.6-fold) and promoted the gene expression of P63 mRNA (increased by 1.4-fold). Furthermore, we found that the addition of the mineralocorticoid receptor antagonist Spironolactone inhibited the increase in the number of ionocytes (decreased to the level of the control group) and suppressed the gene expression of P63 (similarly decreased to the level of the control group). We also provided evidence for gr, mr, and p63 localization in epidermal cells. At the transcript level, mr mRNA is ubiquitously expressed in gill sections and present in epidermal stem cells (cells labeled with p63), supporting the antagonism and functional assay results in larvae. Our results confirmed that cortisol stimulates ionocyte differentiation in tilapia through the MR, rather than the GR. Therefore, we provide a new direction for investigating the dual action of osmotic regulation and skin/gill epithelial development in tilapia, which could help resolve previously inconsistent and conflicting findings.

The utility of p63, CK7, and CAM5.2 staining in differentiating pagetoid intraepidermal carcinomas.

Extramammary Paget disease (EMPD), pagetoid squamous cell carcinoma in situ (PSCCIS), and Paget disease of the breast (PD) are intraepidermal carcinomas with overlapping histopathologic features. CK7 and CAM5.2 stains are frequently utilized to distinguish PSCCIS from EMPD and PD. However, some cases of PSCCIS can stain positively for CAM5.2 and CK7, indicating a potential pitfall with these stains. p63 has been shown to distinguish PSCCIS from EMPD. We assessed p63 staining in PD and compared it to p63 staining of PSCCIS and EMPD. A retrospective search for 15 examples each of PSCCIS, EMPD, and PD with remaining tissue in the paraffin block was performed. The diagnosis was confirmed by a board-certified dermatopathologist and immunostaining for p63, CK7, and CAM5.2 was performed. Staining >55% was scored as positive. Staining <55% was scored as negative and an approximate percentage of positive cells was recorded. Diffuse nuclear expression for p63 was detected in 100% (15/15) of PSCCIS cases, 0% (0/15) of PD cases, and 0% (0/15) of EMPD cases. CK7 and CAM5.2 stains were positive in 100% of PD. CAM5.2 was positive in 100% of EMPD and CK7 was positive in 93% of EMPD. CAM5.2 was positive in 0% of PSCCIS biopsy specimens, but partial staining was seen in 20%. CK7 was positive in 13%, but partial staining was seen in 47%. p63 immunostaining is a highly sensitive and specific method for differentiating between PSCCIS and PD or EMPD. While CAM5.2 and CK7 are also useful ancillary stains in this differential diagnosis, false-positive and false-negative staining occurs with these two markers.

ROLE OF EXPRESSION OF P63 AND CALPONIN IN PROSTATIC BIOPSIES

Background: An increased number of mortality and morbidity in elderly men is caused due to prostatic diseases all over the world. An increase in mass screening programmes conducted with the help of Prostate Specic Antigen (PSA) blood test along with Digital rectal examination (DRE) and imaging studies has resulted in an upsurge in the diagnosis of prostatic cancer which is markedly noted in the recent decades. Material And Methods: A total 100 cases of formalin xed parafn embedded histological sections of various prostatic lesions including benign prostatic hyperplasia, prostatic intraepithelial lesion and prostate carcinoma were studied. Out of 100 cases, 55 cases were benign prostatic hyperplasia, 10 cases of prostatic intraepithelial lesion and 35 cases were of prostate adenocarcinoma. All the sections were stained with Hematoxylin and Eosin stain and then immunohistochemical staining of p63 and Calponin was performed on all the cases (gure 6- 12). The parameters used to analyse the expression of both p63 and calponin were - Pattern of staining, Percentage of stained cell, Intensity of staining reaction. Total 100 cases i Results: ncluded in this study, out of which 55 (55%) cases were benign prostatic hyperplasia, 10 (10%) cases of prostatic intraepithelial neoplasia, and 35 (35%) cases were prostatic adenocarcinoma, majority of specimens (78%) were TURP followed by trucut biopsy (20%), Suprapubic prostatectomy (2%). The proportion of Transurethral resection of prostate (TURP) was signicantly higher as compared to the proportion of Trucut biopsy and supra pubic prostatectomy (SPP) (p&lt;.001). Expression of calponin in various prostatic lesion. On analysing expression of proliferation marker calponin, in BPH it was expressed in only 52 cases out of 5 cases (94.5%) and 3 cases (8.6%) were negative. Our study concluded that the proportion of Benign prostatic Conclusion: hyperplasia was signicantly higher as compared to the Prostatic intraepithelial neoplasia and Prostatic adenocarcinoma. Immunohistochemical staining by p63 is diagnostically reliable in identifying basal cells in prostatic needle biopsies and TURP specimens. p63 is a valuable tool with high sensitivity in differentiating BPH from prostatic carcinoma. The decreased or absent stromal staining of calponin which indicates a reactive stroma in malignancy can be used alone or in conjunction with p63 for conrmation of diagnosis of prostatic carcinoma

Metastases to the kidney from primary lung cancer : clinicopathological analysis of six cases in a single center

ObjectivesCancer metastasis to the kidney is a rare event. We retrospectively analyzed clinicopathologic characteristics in 6 cases of diagnosed renal metastases from primary lung cancer. We also provide clinical follow-up data and brief review of the literature.MethodsImmunohistochemistry was used to evaluate the expression of TTF-1, NapsinA, CK7, CK(AE1/AE3), P63, P40, CgA, PAX-8, GATA3 and Ki-67 in primary tumor and metastases. Additionally, the clinical characteristics, imaging features, diagnosis, and treatment were analyzed.ResultsWith the help of immunohistochemistry and combined clinical history, we found four cases were lung adenocarcinomas, one case was lung squamous cell carcinoma, and the other case was lung small cell carcinoma metastases to the kidney.The patients were all male by gender and had a mean age of 62 years, and metastasis to the left kidney were more universal. Most of the tumors histological grade originating from the lung were poorly-moderately differentiated, and the time to metastasis to the kidney was relatively short for squamous lung cancer and small cell lung cancer, while the time to metastasis for lung adenocarcinoma was related to its degree of differentiation. Overall, we found the prognosis of lung cancer patients with renal metastases were poor especially with multi-site metastases.ConclusionsDistinguishing primary and secondary tumors of the kidney is essential to guide treatment and prevent unnecessary surgery, so clinical information, radiology, histological correlation of the primary tumor, and immunohistochemical findings help the pathologist determine correct diagnosis.

Bladder Cancer Presenting with Cutaneous Metastasis: Case Report

Advanced bladder tumor rarely metastasize to the skin. This is a result of intraperitoneal spread at least microscopically, determining a poor prognosis. We report 73 year old man who presented with skin lesion from pT2 urothelial carcinoma of the bladder. Skin biopsy revealed features of infiltrating urothelial carcinoma. In immunohistochemistry, proliferation expresses CK7 diffusely and CK20 more heterogeneously. It also expresses GATA3 expression of P63. Abdominal-pelvic CT reveled a focal thickening of the bladder dome on the right side of the body, extending over 6 cm and with a maximum thickness of 18 mm, with bilateral pyelocalic dilatation. Patient was referred to oncology for platinum-based chemotherapy. He had responded well to chemotherapy with stable lesions. In any patient followed for a bladder cancer, an atypical skin lesion should raise the alarm and make the patient seek a dermatological opinion for suspicion of a skin metastasis despite its rarity.

Correlation of HER2 With p53 and p63 in Urothelial Bladder Carcinoma.

Urothelial carcinomas account for the majority of all primary bladder cancers, making bladder cancer the second most frequent genitourinary malignancy after prostate cancer.Bladder cancer risk rises with age and most of them return after resection due to their multifocal distribution, and they often develop in superficial locations. Like many other cancers, bladder carcinoma is associated with a few tumor markers that have been evaluated in the past. They include p53, p63, and HER2. This study was conducted on 88 patients suspected of urinary bladder carcinoma. This prospective study was done at the Department of Pathology, Osmania General Hospital, Hyderabad from August 2017 to July 2019. Of the 88 patients, 76 were diagnosed with bladder carcinoma and the remaining 12 were non-neoplastic. The primary neoplastic lesions of the urinary bladder were predominantly seen in patients older than 40 years and were found to be statistically significant (p<0.01). Of the 34 cases of high-grade papillary urothelial carcinoma (PUC), 26 (76.47%) were males, eight cases (23.53%) were females, while among the 25 cases of low-grade PUC, 20 cases (80%) were males, and five cases (20%) were females. In seven cases of squamous cell carcinoma, six cases (85.71%) were males and only one case (14.29%) was female. Of the two cases of adenocarcinoma, male and female gender accounted for one case each (50%). The two cases of papillary urothelial neoplasm of low malignant potential were males in the study. On the whole, the primary urinary bladder lesions are more predominant in the males (77.63%) than the females (22.37%). Overexpression of p53 is negatively connected to p63 expression, and HER2 and p53 were strongly associated with high tumor grade in urothelial carcinoma.

Association of human papillomavirus (HPV), p16, p53 and p63 expression with non-bilharzia-associated squamous cell carcinoma of the bladder and algorithm construction for histopathological grading prediction.

To investigate the expression of human papillomavirus (HPV), p16, p53, and p63 in non-schistosomiasis-related squamous cell carcinoma of the bladder and to develop an accurate and automated tool to predict histological classification based on clinicopathological features. Twenty-eight patients with primary bladder pure squamous cell carcinoma who underwent cystectomy or transurethral resection of bladder tumor (TURBT) for bladder cancer between January 2011 and July 2017 were evaluated. Clinical data and follow-up information were obtained from medical records. Formalin-fixed, paraffin-embedded surgical specimens were used for immunohistochemical staining for p16, p53, and p63. Human papillomavirus detection was evaluated by PCR. Statistical analysis was performed, and statistical significance was set at p<0.05. Finally, decision trees were built to classify patients' prognostic features. Leave-one-out cross-validation was used to test the generalizability of the model. Neither direct HPV detection nor its indirect marker (p16 protein) was identified in most cases. The absence of p16 was correlated with less aggressive histological grading (p=0.040). The positive p16 staining detection found only in pT1 and pT2 cases in our sample suggests a possible role for this tumor suppressor protein in the initial stages of bladder squamous cell carcinoma. The decision trees constructed described the relationship between clinical features, such as hematuria/dysuria, the level of tumor invasion, HPV status, lymphovascular invasion, gender, age, compromised lymph nodes, and tumor degree differentiation, with high classification accuracy. The algorithm classifier approach established decision pathways for semi-automatic tumor histological classification, laying the foundation for tailored semi-automated decision support systems for pathologists.

Acral BRAF‐mutated tubular adenoma should be distinguished from HPV42‐related digital papillary adenocarcinoma

Acral <i>BRAF</i>‐mutated tubular adenoma should be distinguished from <scp>HPV42</scp>‐related digital papillary adenocarcinoma

Global status of research on cutaneous squamous cell carcinoma and its programmed cell death: Bibliometric and visual analysis from 2012 to middle 2022.

This study was aimed to analyze the research status and development trend of cutaneous squamous cell carcinoma (CSCC), focusing on the field of programmed cell death of CSCC, and providing suggestions for the research of CSCC. The publications related to CSCC and CSCC programmed cell death were searched in the Web of Science Core Collection (WOSCC) database, and timespan was set from 2012 to middle 2022. Research trends, authors, major country collaborations, research institutions, representative journals, publishers, and keywords were analyzed with CiteSpace and VOSviewer. After screening, a total of 3656 publications on CSCC and 156 publications on CSCC cell programmed death were obtained. The number of published articles increased gradually with the years. The United States ranked first in terms of the number of published papers. Research in this field had focused on dermatology. Most of the institutions in both regions were from European and American countries. Harvard University was the most prolific institution. Wiley was the most prolific publisher. The popular keywords for programmed cell death in CSCC were cutaneous squamous cell carcinoma, diagnosis, pd 1, head, nivolumab and risk. Keywords in CSCC field were divided into 7 clusters: cutaneous squamous cell carcinoma, sentinel lymph node biopsy, skin cancer, B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF) inhibitor and human Papillomaviruses, and P63 expression. Squamous cell carcinoma, cancer, head and expression were the most popular keywords. The popular keywords for programmed cell death in CSCC were cutaneous squamous cell carcinoma, diagnosis, pd 1, head, nivolumab and risk. This study analyzed the research status of cutaneous squamous cell carcinoma and programmed cell death from 2012 to middle 2022. Understanding research status and hotspots can help scholars, countries and policymakers to better understand the background and research frontier of CSCC, and guide further research directions.

Abstract 3436: Reduced p63 expression is linked to a low density of regulatory T-cells and unfavorable prognosis in muscle-invasive urothelial carcinoma of the bladder

Abstract Background: Tumor protein 63 (p63) is a transcription factor of the p53 gene family which is regularly expressed in the normal urothelium. Recently proposed RNA expression based molecular classifiers of bladder cancer identified high p63 expression as a component of a basal/squamous subtype linked to poor patient prognosis. The interplay between p63 expression status and the anti-tumor immunity in bladder cancer is unknown. Design: To assess the prognostic impact of p63 expression and the relationship between p63 and the immune tumor microenvironment we have stained tissue microarrays containing more than 2300 urothelial bladder carcinomas with 22 antibodies (i.e., p63, CD3, CD8, CD4, FOXP3, CD20, CD68, CD163, CD11c, TIM3, PD-L1, PD-1, CTLA-4, panCK, Ki-67, CD31, Vimentin, HLA-DRa, Myosin-11, Desmoglein 3, PAX-8, CDH16) using conventional brightfield and multiplex fluorescence immunohistochemistry (BLEACH&amp;STAIN). A framework of several neuronal networks for image analysis were used. Spatial immune parameters were compared with histopathological parameters and overall survival data. The area under (tAUC) time-dependent receiver operating characteristic curves was used to compare the prognostic relevance of different prognostic markers. Results: Nuclear p63 staining was seen in all cells of normal urothelium and in all pTaG2 tumors, mostly at high levels. The rate of p63 positive cases and the staining intensity was lower in pTaG3 tumors (93.2%, p&amp;lt;0.0001 for pTaG3 vs pTaG2) and markedly lower in pT2-4 carcinomas (83.5%, p=0.0120 for pT2-4 vs. pTaG3). A low p63 expression was linked to a low density of T-helper cells (p=0.044) and regulatory T-cells (p=0.0053) localized in the intraepithelial tumor component as well as in the stroma, while all other analyzed T-cells and macrophages subsets where unrelated to p63 expression. Within pT2-4 carcinomas, low p63 expression was linked to nodal metastasis (p=0.0028) and overall survival (p=0.0005). The association of p63 loss with survival was independent of pT and pN (p=0.0109). The predictive performance of intraepithelial CD8+ cytotoxic T-cells (tAUC: 0.70) was even higher than the predictive performance of p63 expression (tAUC: 0.57, p=0.0017). Conclusion: In summary, our data show that p63 is downregulated in a fraction of urothelial neoplasms that are associated with a particularly poor prognosis and a low density of T-helper and regulatory T-cells. The even higher predictive performance of intraepithelial CD8+ cytotoxic T-cells underlines the strong prognostic role of the immune tumor microenvironment in muscle invasive bladder cancer. Citation Format: Henning Plage, Sebastian Hofbauer, Kira Kornienko, Paul G. Bruch, Sarah Weinberger, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Martina Kluth, Maximilian Lennartz, Tim Mandelkow, Elena Bady, Niclas C. Blessin, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Steffen Hallmann, Stefan Koch, Nico Adamini, Sarah Minner, Ronald Simon, Guido Sauter, Tobias Klatte, David Horst, Thorsten Schlomm, Henrik Zecha. Reduced p63 expression is linked to a low density of regulatory T-cells and unfavorable prognosis in muscle-invasive urothelial carcinoma of the bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3436.

PD35-11 EPITHELIAL DIFFERENTIATION IS ALTERED IN URETHRAL LICHEN SCLEROSUS

You have accessJournal of UrologyCME1 Apr 2023PD35-11 EPITHELIAL DIFFERENTIATION IS ALTERED IN URETHRAL LICHEN SCLEROSUS Ethan Richmond, Caleb Sokovich, Wei Huang, Chad Vezina, and Matthew D Grimes Ethan RichmondEthan Richmond More articles by this author , Caleb SokovichCaleb Sokovich More articles by this author , Wei HuangWei Huang More articles by this author , Chad VezinaChad Vezina 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.0000000000003333.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lichen sclerosus (LS) is an idiopathic chronic inflammatory disease of the genital epithelium resulting in fibrosis and severe urethral stricture disease in affected men. Epithelia are subdivided into basal, intermediate, and superficial cell layers which are defined by protein expression patterns. Loss of basal cell differentiation is a feature of other fibrotic disorders but have not been studied in the urethra or LS. The proteins keratin 5 (KRT5) and p63 have been reliably established as basal cell markers in urothelium. Therefore, we hypothesized that urethral epithelium affected by LS would have decreased basal expression of KRT5 and p63. METHODS: Urethral tissue after urethral biopsy or urethroplasty was examined by a genitourinary pathologist to determine LS status. Using multiplexed fluorescent immunohistochemistry, KRT 5, CD45, p63, and DAPI were stained and three epithelial regions imaged per specimen. Using QuPath analysis software, the epithelium was segmented into basal and luminal compartments and relative abundance (RA) of each primary antibody was measured (number of positive cells/number of nucleated cells). RA was compared with respect to LS status and basal/luminal compartment using a Mann-Whitney U test. RESULTS: We analyzed 14 slides (n=7 LS, n=7 non-LS). The RA of KRT5 and p63 positive cells were similar with respect to epithelial versus basal compartment and LS status. However, we noted a change in patterning such that LS tissues displayed a shift of KRT5 and p63 expression from the basal to the luminal epithelium. To quantify this, we compared the ratio of basal to luminal KRT5 and p63 expression and dichotomized this as “basal predominant” (>1) or “luminal predominant” (<1). LS urethra were significantly more likely to have luminal predominant expression of p63 and KRT5 compared to non-LS (p=0.03, p=0.03) (Figure 1). CONCLUSIONS: We identified a change in expression pattern of basal epithelial markers (KRT5 and p63) in LS urethra. Altered epithelial differentiation is seen in other inflammatory diseases and may be implicated in the pathogenesis of LS. Source of Funding: K12DK100022NIH/NIDDK © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e978 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ethan Richmond More articles by this author Caleb Sokovich More articles by this author Wei Huang More articles by this author Chad Vezina More articles by this author Matthew D Grimes More articles by this author Expand All Advertisement PDF downloadLoading ...

The High Presence of HCMV pp71 Proteins, Correlate with P63 Expression in Pancreatic Cancer Tumor Tissue.

The purpose of this retrospective investigation was (1) to screen the existence of HCMV in pancreatic cancer tissues in relation to the histopathological grading system of such tumor tissues. (2) To evaluate the expression of the (P63) tumor suppressor gene in these tissues. (3) To find out the impact of the coexistence of (HCMV) along with the p63 on the occurring histopathological alterations. The current retrospective cohort study included 35 paraffinized pancreatic tissues from the archives of major hospitals and numerous private histopathological laboratories from 2015 to 2020. (Twenty-five pancreatic carcinomatous tissues and 10 biopsies from seemingly normal pancreatic tissues were examined). Tissue slices from the desired tissue blocks were subjected to the immunohistochemistry (IHC) technique to detect Human Cytomegalovirus pp71 and tumor suppressor P63 proteins with aid of monoclonal primary antibodies. The HCMV pp71 proteins were found in 92% (23 out of 25) of pancreatic tumor tissues, while it was in two (20%) of healthy pancreatic tissues. in comparison, the p63 proteins were found in 76% (19 out of 25) of tumor tissues and in four (40%) of their correlative healthy tissues. The increased expression of HCMV in malignant pancreatic tissue may indicate its primary or secondary role in the emergence of this type of cancer, whereupon HCMV inactivation may be useful in the treatment of this type of cancer. On the other hand, p63's high levels of expression in malignant pancreatic tumors reflect either an oppressive function or an unfortunate mutation that prevents it from functioning.