Markers P63 Research Articles

Expression of Lymphoid Enhancer-Binding Factor 1 in Cancer-Associated Fibroblasts Mediates Tumor Growth and Transdifferentiation Toward Squamous Cell Carcinoma in Human Breast Cancer.

Cancer-associated fibroblasts (CAFs) play a significant role in human breast cancer as a major stromal component. While their role in promoting cancer proliferation and malignancy through interaction with cancer cells in the tumor microenvironment is known, the exact mechanisms behind this interaction are not fully understood. Our study reveals that lymphoid enhancer-binding factor 1 (LEF1), a central transcription factor for Wnt/β-catenin signaling, is expressed in experimentally generated tumor-promoting CAFs (exp-CAFs) as well as in CAFs from breast cancer patients, particularly those with a poor prognosis. Notably, LEF1-expressing CAFs are prevalent in the stroma of squamous cell carcinoma (SCC), an aggressive metaplastic breast cancer subtype with a limited understanding of its development. To investigate the functional importance of LEF1 expression in CAFs, we depleted LEF1 in the exp-CAFs and subcutaneously implanted them along with breast ductal carcinoma MCF10DCIS.com cells into immunodeficient mice. Depleting LEF1 resulted in reduced xenograft tumor growth, accompanied by decreased cancer-cell proliferation and angiogenesis in the tumors. Additionally, we observed a significant reduction in the expression of SCC markers p40 (ΔNp63) and cytokeratin 5/6 in the xenograft tumors when LEF1 was depleted in the exp-CAFs. Furthermore, we identified 13 genes, none of which are established downstream genes of the Wnt/β-catenin pathway, that exhibit expression patterns similar to LFE1 in our cultured fibroblasts. In summary, our findings suggest that LEF1 expression contributes to the tumor-promoting abilities of breast CAFs and that LEF1-expressing CAFs may drive transdifferentiation toward SCC, possibly through a pathway independent of the canonical Wnt/β-catenin signaling.

Exploring the Active Constituents of Andrographis paniculata in Protecting the Skin Barrier and the Synergistic Effects with Collagen XVII.

Andrographis paniculata is mainly used to treat skin inflammations, wounds, and infections. In this study, Andrographis Herba, the aerial part of the plant, was proven to increase the viability of UVB-damaged HaCat cells and reduce reactive oxygen species levels. The chemical composition of Andrographis Herba extract (AHE) was analyzed using UPLC-Q-TOF-MS, and diterpene lactones were identified as its primary constituents. Then, the fraction of diterpene lactones was prepared and exhibited similar effects to AHE. AHE, its diterpene lactones component, and its representative constituent andrographolide all decreased the expression of IL-1β, IL-6, and CDKN1A. Furthermore, the protective effects of AHE and its active ingredients on UVB-damaged epidermal stem cells were investigated. Notably, the combined treatment with andrographolide and collagen XVII enhanced the viability of UVB-damaged epidermal stem cells, increased the expression of stemness markers integrin β1 and p63, and decreased the expression of the differentiation marker keratin 10. This combination demonstrated significant synergy in maintaining skin homeostasis, which provides evidences for the development of skin-protective products.

Vulvar Lactating Adenoma: A Rare Representation of Mammary-Like Anogenital Glands.

Mammary-like anogenital glands are considered a normal constituent of the anogenital area. These glands can have epithelial components with eccrine or apocrine features. They often undergo transformation into mammary-like lesions, including lactational changes that occur during pregnancy and the breastfeeding period. When they form mass lesions they are referred to as lactating adenomas. Their appearance can clinically be mistaken for other more common benign and malignant entities in the vulva. We describe a vulvar lesion in a 26-year-old woman. The lesion was excised and determined by histologic examination to be a benign lactating adenoma. Subsequent immunohistochemistry was performed. The epithelial cells were positive for GATA3 and estrogen receptor while negative for PAX8, supporting mammary-like differentiation. The myoepithelial markers p63 and calponin were positive at the periphery of the ducts, supporting the benign nature of this lesion.

Abstract B045: Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells

Abstract Background: Breast cancer is the most frequently diagnosed cancer, and the second leading cause of cancer-related death in women. Most breast cancer related deaths are due to metastasis to distant organs. The primary tumor and secondary site microenvironments provide cues that activate cancer cell plasticity programs such as those associated with invasion and stemness, which are needed for dissemination and the establishment of metastatic foci. We demonstrated that in the primary tumor >50% of cancer stem cells (CSCs) co-express the invasive phenotype marker, MenaINV. Given that ∼80% of newly arriving circulating tumor cells to the lung are CSCs, we questioned if extravasation of CSCs to the lung is mediated solely by MenaINV or if other compensatory pathways are at play. We describe here our discovery that CSCs require MenaINV for extravasation to the lung parenchyma. Methods: To visualize breast cancer stem cell extravasation into the lung parenchyma, we expressed the stemness biosensor, SORE6, in MDA-MB-231 breast cancer cells. SORE6 is composed of 6 tandem repeats of the Sox2 and Oct4 response elements placed upstream of destabilized Green Fluorescent Protein (GFP). To distinguish CSCs (green) from non-CSCs, all cells were also labeled with red tracker dye. Breast cancer cells transduced with lenti-SORE6 were FACS sorted into low SORE6-GFP cells (non- CSCs) and high SORE6-GFP (CSCs). Activation of the stem program in FACS sorted cells was validated by qRT-PCR analysis of the widely used stem cell markers CD44, CD133, P63, ALDH1, Sox9, Sox2, and Oct4. The extravasation efficiency of non-CSCs vs. CSCs was assessed in vitro by the extravasation directed transendothelial migration (eTEM) assay, which mimics extravasation by allowing migration of cancer cells through endothelial cells cultured on a porous membrane, as well as in vivo, by intravital imaging. The impact of MenaINV on CSC extravasation was evaluated through CRISPR/Cas9 mediated knockout of MenaINV. In in vivo experiments, a 50/50 mixture of non-CSCs and CSCs expressing either wild type or knocked-out MenaINV were injected into the vasculature of Rag2-/- MacBlue mice. in vivo visualization of cancer cell extravasation was accomplished by implantation of an indwelling lung imaging window prior to the injection of cancer cells, followed by high-resolution intravital imaging. Results: CSCs exhibit significantly higher expression of all stem cell markers and express more MenaINV compared to non-CSCs. Moreover, CSCs show significantly better transendothelial migration in eTME assay (p=0.03) and lung extravasation (p<0.0001) in intravital imaging, compared to non-CSCs. Finally, knocking out MenaINV reduced lung extravasation to the levels comparable to non-CSCs. Conclusions: This study highlights the crucial role of MenaINV in facilitating lung metastasis by CSCs. Thus, targeting MenaINV may represent a promising therapeutic strategy to reduce metastatic burden in breast cancer patients. Citation Format: Mohd Nauman, Yookyung Jung, Burcu Karadal-Ferrena, Camille Duran, Madeline Friedman-DeLuca, Nicole Barth, Prachiben Patel, John Condeelis, David Entenberg, Maja H. Oktay. Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B045.

The Diagnostic And Prognostic Role Of Immunohistochemistry In Head & Neck Tumours Along With Radiological & Histopathological Study

Background: Head & neck tumors are defined as benign, premalignant & malignant tumors above the clavicles, except the tumors of brain, spinal cord and esophagus. This includes tumors of paranasal sinus, nasal cavity, salivary glands, thyroid and upper aerodigestive tract, lymph nodes of neck, skin & its appendages, soft tissue tumors, midline carcinomas & metastatic tumors. Ultrasonography, computed tomography, magnetic resonance imaging were the modalities for initial evaluation. Histopathology was the GOLD standard for diagnosis followed by IHC for confirmation because correct diagnosis & cancer subtyping of the neoplasia is essential for successful therapy and prognosis. Materials & Methods: Study was conducted for 2 years with 100 specimens of head & neck tumors. Thorough Radiological investigation was done followed by Histopathological examination including biopsy & whole resected specimens. IHC markers appropriate to the histopathological diagnosis was done. Results: Among 100 patients of head & neck tumors, most belonged to 5th decade with male: female ratio of 2.3:1. Squamous cell carcinoma (66%) followed by Lymphoma(11%), tumors of salivary glands(8%), metastatic lymphadenopathy(7%) were the lesions present. SCC was most common carcinoma in oral cavity, larynx, nasopharynx and ear with positive p63 marker. 1 case of Basal cell carcinoma with positive BerEP4 & in NHL, salivary gland tumors & Metastatic lymphadenopathy panel of IHC were included for confirmation. Conclusion: Immunohistochemistry has a diagnostic & prognostic role in Head & Neck tumors along with Radiological & histopathological examination.

Multi-omics profiling the genomic origin and adeno-squamous transdifferentiation mechanism in adenosquamous carcinoma.

e20032 Background: Adenosquamous carcinoma (ASC) represents a rare subtype (0.4-4%) of non-small-cell lung cancer (NSCLC), with its multi-omic landscape yet to be fully elucidated. This study aims to explore the genomic origins and molecular mechanisms of adeno-squamous transdifferentiation in lung ASC through comprehensive multi-omics. Methods: We analyzed 69 samples from 31 lung ASC patients, including 16 FFPE samples with microdissected paired adenocarcinoma (AC) and squamous cell carcinoma (SCC) components, and additionally, incorporated pure Lung Adenocarcinoma (LUAD, n = 51) and pure Squamous Cell Carcinoma (LUSC, n = 28) for comparative analysis. Techniques included whole-exome sequencing, whole-genome methylation profiling, transcriptomics, single-cell RNA sequencing, and spatial transcriptomics. Results: AC and SCC exhibited similar genomic mutation landscapes, indicating a common genomic origin for AD and SCC. The SCC component in ASC displayed higher homologous recombination repair deficiency (HRD) and a notable 16p13.3 deletion in 31% of ASC transformations. Differential methylation analysis highlighted TP63's lower methylation in SCC, correlating negatively with RNA expression (P < 0.001), indicative of methylation-driven regulation. DMR binding motif analysis revealed hypo-methylation in p63, p53, and AP-1 motifs, and hyper-methylation in MKX2-1, FOXA1/2 motifs (P < 0.05). The transition from AC to SCC in ASC was marked by upregulated MYC signaling, proliferation, and DNA repair, with EMT changes not methylation-driven. Metabolic pathways were upregulated, and EMT/KRAS pathways downregulated in SCC compared to LUSC. Immune infiltration in LUAD-AC-ASC transformation showed an initial increase and subsequent decrease, transitioning from immune-infiltrated and immune-excluded to immune-desert phenotypes. IL36G of the IL1 family was highly expressed in the SCC component and strongly correlated with the squamous marker P63 (Pearson r = 0.72, P < 0.001). Single-cell RNA-seq data demonstrated a significant increase in IL36G in squamous cells of lung ASC. In vivo models showed adeno-squamous differentiation in adenocarcinoma cells expressing IL36G. Conclusions: This study provides a comprehensive multi-omic insight into the complex genomic and epigenomic landscape of ASC, highlighting a shared genomic origin and distinct molecular mechanisms in the adeno-squamous transdifferentiation process. The findings, particularly the role of TP63 methylation and IL36G expression in the SCC component, offer novel insights into the molecular heterogeneity of ASC. These results not only deepen our understanding of ASC but also pave the way for future investigations into targeted therapies that could potentially exploit these molecular distinctions for more effective treatment strategies in ASC.

Abstract PO2-20-08: Metastatic Lung Cancer Masquerading as Metaplastic Breast Cancer

Abstract Background Metaplastic breast carcinoma (MBC) is an unusual malignancy that presents a diagnostic challenge due to the presence of varying cytomorphologies that can be seen in benign and malignant tumors. We posit that metastatic disease to the breast, itself an uncommon entity, should be considered in the differential diagnosis of MBC. We report a unique case of metastatic non-small cell lung cancer (NSCLC) with an actionable driver mutation that presented as a symptomatic breast mass and was initially considered to represent MBC. Report A 74-year-old woman with a 22-pack-year smoking history presented to the breast clinic complaining of a palpable left breast mass that had grown rapidly over the previous month. Ultrasound-guided biopsy of the breast mass revealed high-grade poorly differentiated carcinoma. On immunohistochemical (IHC) evaluation, tumor cells were strongly positive for epithelial markers CAM5.2 and keratin AE1/AE3, weakly positive for breast cancer (BC) marker TRPS1, and negative for BC marker GATA3, squamous cell carcinoma markers p63 and CK5, and melanoma marker SOX10. Metaplastic triple-negative BC was diagnosed. However, concern remained for metastasis from a non-breast primary tumor due to the patient’s constitutional symptoms such as fatigue, night sweats, and 60-pound unintentional weight loss over the previous year, as well as interval development of a suspicious nodular rash on her right upper extremity. An FDG-PET-CT was obtained and demonstrated a hypermetabolic left lung consolidation (maximum SUV 33.3) measuring 7.2 cm, pulmonary nodules, and extensive thoracic lymphadenopathy. Due to rising suspicion for a primary lung tumor, additional IHC staining on the original breast biopsy was requested. Tumor cells were positive for TTF-1, consistent with metastatic lung adenocarcinoma. Tissue next-generation sequencing (NGS) identified a MET exon 14 skipping mutation, making the patient eligible for treatment with capmatinib, a first-line kinase inhibitor targeted therapy for metastatic NSCLC. At the time of this report, the patient has completed a course of palliative stereotactic body radiation therapy to the left lung and started capmatinib 400 mg BID, which she is tolerating well. Her first restaging scans after treatment initiation will be obtained one week after the time of this report. Conclusion In the absence of heightened suspicion for a non-breast primary tumor, our patient had initially received a diagnosis of triple-negative MBC. This diagnosis would have exposed her to increased treatment-related toxicities from cytotoxic chemotherapy and poor prognosis from MBC without survival benefit from targeted therapy for NSCLC. We advocate for the use of supplemental body imaging, expert and comprehensive IHC evaluation, and multidisciplinary discussion in cases of MBC and undifferentiated breast malignancy to facilitate accurate diagnosis and treatment. Citation Format: Tanmayi Pai, Marte Wasserman, Jason Lewis, Miglena Komforti, James Jakub, Augustine Lee, Yanyan Lou, Pooja Advani, Rohit Rao. Metastatic Lung Cancer Masquerading as Metaplastic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-20-08.

Abstract 222: Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments

Abstract The risk of relapse or recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is high despite the combination of surgery and radiochemotherapy, responsible for high toxicity. It is crucial to develop new therapeutic strategies and to identify patients likely to benefit from these treatments. Patient-Derived Tumor Organoids (PDTO) are three-dimensional multicellular structures derived from patient tumor samples and faithfully reproduce the histological and molecular characteristics of the original tumor. A growing body of research indicates that PDTO may predict the clinical response, representing a major opportunity for the development of new therapeutic strategies and precision medicine. The purpose of this translational study is to generate PDTO from HNSCC patients to evaluate their predictive value. PDTO were obtained after dissociation of tumor specimen of HNSCC patients through the setup of the clinical study ORGAVADS (NCT04261192). Tumor cells were embedded in extracellular matrix and cultured in specific medium. Histological and immunohistochemical characterizations were performed to validate the resemblance between PDTO and their original tumor. Response of PDTO to chemotherapy, radiotherapy and innovative therapies were evaluated by live-cell imaging and viability assays. The culture conditions were optimized to improve the success rate of PDTO establishment which now exceeded 50%. Twenty-one PDTO lines have been established and showed histological characteristics close to the original tumor. Expression of immunohistochemical tumor markers p53, p40, p63, and p16 were similar in tumors and paired PDTO. Functional assays were performed on 15 PDTO to analyze the response to treatments (cisplatin, olaparib, X-Rays) and heterogeneity of response was observed between the models. When clinical response was available, PDTO derived from good responders showed high sensitivity to treatments while PDTO from bad responders showed low sensitivity to treatments. Interestingly, two PDTO lines derived from HPV+ oropharyngeal tumors displayed very high sensitivity to cisplatin, matching with the patient's profile and response. Our results showed feasibility to derive PDTO from HNSCC and to perform functional assay to assess their response to different treatments. The first studies of correlation between PDTO and patient responses are promising and will be further investigated in more patients. This will allow to demonstrate the predictive value of PDTO from HNSCC with the purpose to develop a clinically compatible predictive functional assay. Citation Format: Marion Perréard, Vianney Bastit, Lucie Lecouflet, Guillaume Desmartin, Romane Florent, Corinne Jeanne, Juliette Thariat, Emmanuel Babin, Laurent Poulain, Louis-Bastien Weiswald. Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 222.

A comparative study of morphological and Immunohistochemical expression of P40 and P63 immunomarkers in squamous cell carcinoma and adenocarcinoma lung

Background: Lung cancer is the leading cause of cancer related death worldwide and accounts for 28% of all cancer mortality and around 1.8 million new cases were diagnosed in 2012. The morphological distinction between pulmonary adenocarcinoma (ADC) and squamous cell carcinomas (SCC) is sometimes difficult, mainly in cases of poorly differentiated tumors or when degenerative changes, necrosis and crushing may obscure the cell characteristics. p63 is a homologue of the p53 tumour suppressor gene that is responsible for proliferation and differentiation of epithelial progenitor cells. p40 is consistently the predominant isoform expressed in squamous cell carcinoma; thus, it offers improved specificity for diagnosing squamous cell carcinoma. Material and Methods: This is a prospective and observational study conducted in the Department of Pathology, Tertiary care Teaching Hospital over a period of 1 year. Primary lung carcinoma cases included with unequivocal morphological diagnosis irrespective of age, gender and nature of biopsy material (endoscopic biopsy/ needle core biopsy / resected specimen). Cases diagnosed as Small cell carcinoma of lung, as metastatic lung cancers, poorly differentiated NSCLC-NOS and with inadequate material for IHC study were excluded from present study. Results: In the present study, a total of 150 patients were included out of which 112 (74.6%) were males and 38 (25.4%) were females. In our study, most of the patients were >61 years i.e., 63 out of 150 (42%), followed by 51-60 years, i.e., 40 out of 150 (26.7%). All 8 cases of well differentiated Adenocarcinoma were positive for P40 and 5 cases showed P63 expression. Out of 13 cases of moderately differentiated Adenocarcinoma, 3 cases were positive for P40 and 10 cases were positive for p63 marker. Out of 30 cases of well differentiated Squamous cell carcinoma 15 cases were P40 positive and 15 cases were P63 positive. All 53 cases of moderately differentiated Squamous cell carcinoma showed positive P40 and P63 expression.

A study on immunohistochemical expression of HER2/Neu and p63 and its association with grade and invasiveness in case of bladder carcinoma.

Bladder cancer is a global disease, ranks as the fourth most prevalent cancer. The incidence and prevalence increase with age. Grade and aggressiveness have been found to be related with different genetic expression and mutation. To evaluate any relation of grade and invasiveness of urothelial cancer with varied expression of immune histochemical marker p63 and her2/neu. The present study was a hospital based prospective cross-sectional study. This Study was conducted from July 2021 to April 2023 in the Urology department of a tertiary care hospital. Total 90 patients undergoing trans urethral resection of bladder tumour (TURBT) were included in this study. It was found that, patients who had decreased p63 expression had high grade in tumours (93.1%) compared to patients who were expressing normal p63 (32.8%) and this was statistically significant (p < 0.0001). Tumours with decreased p63 also appeared to be more invasive, 62.1% were found to be muscle invasive. Tumours with her2 neu expression found to be more aggressive in nature, 85.7% had high grade features and 53.6% were muscle invasive. Our findings suggest that immunohistochemical expression of HER2/neu positive and decreased p63 expression were associated with high grade and invasiveness in case of bladder carcinoma.

The existence of senescent cells in conjunctival epithelium from elderly individuals.

The ocular surface microenvironment changes with aging. However, it remains unclear if cellular senescence influences the ocular surface. We investigated the presence of p16INK4a-expressing senescent cells in healthy human conjunctiva. Clinical and experimental. Healthy conjunctival tissue samples were obtained from middle-aged and elderly subjects. RT-qPCR was performed to assess the expression of senescence markers CDKN2A (p16INK4a) and CDKN1A (p21CIP1/WAF1) and immunostaining was performed to examine the expression of the senescence marker p16INK4a, stem cell markers Ki67 and p63, tight-junction marker ZO-1. Our study involved 19 conjunctival tissue samples (10 elderly and 9 middle-aged), mean age [elderly: 75.8 ± 3.7 years (72-81), middle-aged: 52.7 ± 7 years (38-59)], sex (elderly: 3 men, 7 women; middle-aged: 3 men, 6 women). The expression of p16INK4a was significantly increased at the RNA level in the elderly compared to middle-aged (p < 0.05). Positivity rate of p16INK4a was significantly elevated in the elderly (15.0 ± 7.8%) compared to middle-aged (0.2 ± 0.6%) (p < 0.05). Positivity rate of Ki67and p63 was significantly reduced in the elderly (1.7 ± 1.7% and 16.5 ± 9.5%) compared to middle-aged (3.9 ± 1.8% and 24.7 ± 5.7%) (p < 0.05). ZO-1 expression was reduced in tissue samples showing p16INK4a-positivity but retained in tissue samples in which p16INK4a was undetectable. Senescent cells accumulate with age in the conjunctival epithelium, accompanied by a decrease in Ki67, p63 and ZO-1 expressing cells.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

BackgroundLimbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.MethodsAfter in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.ResultsExpression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.ConclusionThis manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

Expression of p53, p63, p16, Ki67, Cyclin D, Bcl-2, and CD31 Markers in Actinic Keratosis, In Situ Squamous Cell Carcinoma and Normal Sun-Exposed Skin of Elderly Patients

Background: Age and cumulative exposure to ultraviolet (UV) light are primary contributors to skin cancer development. Regulatory proteins within the cell cycle are essential for the homeostasis of squamous epithelium. Methods: This study assessed the expression of immunohistochemical markers p53, p63, p16, Ki67, Cyclin D, Bcl-2, and CD31 in keratinocyte intraepithelial neoplasia (actinic keratosis and squamous cell carcinoma in situ) compared to normal skin. The objective was to distinguish disease-specific changes from those attributable to ageing and sun exposure in elderly skin. Results. Analysis included 22 actinic keratoses (AK), 7 in situ squamous cell carcinomas (SCC), and 8 normal skin biopsies. The mean age was 78.1 years for the AK/SCC group and 73.8 years for controls, with no significant age difference noted between the groups. The AK/SCC group exhibited a higher occurrence of amorphous masses, higher intensity of p53, lower Bcl-2 expression in the epidermis, higher Bcl-2 expression in the dermis, and higher CD31 expression in the dermis, all of which were statistically significant (p &lt; 0.05). Conclusions: The study identifies distinct differences in the presence of amorphous masses and the expression levels of p53, Bcl-2, and CD31 between sun-exposed skin and in situ cutaneous squamous cell carcinomas, including actinic keratoses.

B-376 Performance of Pathological Anatomy Immunohistochemical Markers and Serum Tumor Markers in Prostate Cancer

Abstract Background The gold standard method for diagnosing prostate cancer is systematic prostate biopsy under ultrasound guidance and local anesthesia.Serum tumor marker tests (PSA, Free PSA and testosterone) and confirmatory or classificatory immunohistochemical (IHC) complement the conclusion of the patient’s report. The maximum amount of information regarding the pathological anatomy and markers provided to physicians favors assertive decision-making in the conduct of treatment. However, the results of prostate biopsies after treatment with prostatectomy can differ significantly in the course of the disease. In this context, the authors’ objectives were to analyze the performance of the dosages of these widely used serum markers against the IHC markers of biopsies, as well as their indicators of positive and negative predictive values (PPV and NPV) for prostatic neoplasms. The authors analyzed the level of agreement between pathological prostate biopsy results and immunohistochemical (IHC) testing for 34ßE12, p63, and alpha-methylacyl-CoA racemase (AMACR) to provide final pathological results for prostate cancer, as well as their relationship with data provided by biochemical tests for the markers PSA, Free PSA and Testosterone. Methods Descriptive, retrospective study with a qualitative and quantitative approach, based on data obtained through IHC exams and available in a database of a large laboratory in São Paulo—Brazil. Data collection was based on anatomopathological results of male patients diagnosed with prostate cancer, aged between 40 and 69 years between August 2021 and December 2022 with a total of 1403 analyzes for biopsies using IHC combined markers such as 34ßE12 , p63 and AMACR, and a total of 144 analyzes for combined prostatic serum markers (total PSA, free PSA and testosterone). All analyzes were conducted using the R software version 4.1.0 (R CORE TEAM, 2021) and considered a significance level (α) of 5%. Results The Mann-Whitney test indicated that the distribution of the PSA variable (ng/dL) does not differ statistically between the 34ßE12 groups (W = 822.0; P = 0.224; r = 0.139 , p63 (W = 826.0; P = 0.361 ; r = 0.103), and AMACR (W = 610.0; P = 0.351; r = -0.106). Likewise, the Free PSA marker (ng/dL) did not differ statistically between 34ßE12 (W = 309.5; P = 0.473 ; r = -0.097), p63 (W = 319.5; P = 0.488; r = - 0.093 and AMACR (W = 372.0; P = 0.725; r = 0.048). The results of the ROC curve with PSA (ng/dL) as predictor of the biopsy result with the marker 34ßE12, p63 and AMACR the mean positive predictive values (MPVP) were 0.623 , 0.635 and 0.615 respectively. For Free PSA (ng/dL) with the markers respectively, the VPPM were 0.743, 0.750 and 0.455. As for testosterone, our sample was very small. Conclusion Our findings showed that all markers are important indicators for the diagnosis and monitoring of prostate cancer and that they complement each other. Free PSA showed good PPV for 34ßE12 and p63 markers and low PPV for AMACR.

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.

Pax6-Induced Proliferation and Differentiation of Bone Marrow Mesenchymal Stem Cells Into Limbal Epithelial Stem Cells.

Corneal integrity, transparency, and visual acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by limbal epithelial stem cells (LESCs). The limbal stem cell deficiency is associated with ocular diseases. This study aimed to develop a novel method to differentiate bone marrow mesenchymal stem cells (BM-MSCs) into LESC-like cells using a culture medium and paired box 6 (Pax6) transfection. The LESC-like cells were confirmed using the LESC markers CK14 and p63 and CEC marker CK12. Pax6 induces BM-MSCs to differentiate into LESC-like cells in vitro. Mouse models of chemical corneal burn were obtained and treated with the LESC-like cells. The transplantation experiment indicated that Pax6-reprogrammed BM-MSCs attached to and replenished the damaged cornea through the formation of stratified corneal epithelium. The proliferation and colony formation abilities of Pax6-overexpressing BM-MSCs were significantly enhanced. These findings provide evidence that BM-MSCs might serve as an excellent candidate for generating bioengineered corneal epithelium and provide a new strategy for the treatment of clinical corneal damage.

MIOEPITHELIAL CELLS: STRUCTURE, FUNCTION, AND THEIR ROLE IN NORMAL AND NEOPLASTIC TRANSFORMATION OF THE BREAST

Abstract. Myoepithelial cells (MECs) are characteristic of exocrine glands and are responsible for synthesizing the extracellular matrix, particularly the structural components of the basal membrane. MECs are of epithelial origin and exhibit contractile function, which aids in milk ejection from the ducts during lactation. Scientific literature suggests that MECs act as suppressors of tumors and play an important diagnostic role in mammary gland pathology, especially in distinguishing diff erent neoplastic processes.Research objective: T o elucidate the histological features and immunohistochemical verifi cation of myoepithelial cells in normal breast tissue and their role in neoplastic transformation of the mammary gland.Materials and Methods. The histological examination of myoepithelial cells of the mammary gland was conducted on archival slides from the Department of Histology, Cytology, and Embryology, Danylo Halytsky Lviv National Medical University. Histological sections, stained with hematoxylin and eosin, as well as immunohistochemical markers p63 (Clone 4A4, Masterdiagnostica) and cytokeratin 5/6Ab-2 (D5/16 B4, Thermoscientifi c) for confi rming the myoepithelial origin of the cells, were analyzed under a light microscope (UlabXSP-137TLED) with 10x and 40x objectives and a 10x eyepiece. Photographs were taken using an XCAM 1080P camera.Research Results. The structural units of the mammary gland (MG) are ductal-l obular formations, the terminal portions of which have an alveolar shape and are formed by secretory exocrine cells with underlying myoepithelial cells – basket-like cells. Identifi cation of myoepithelial cells using conventional histological staining techniques such as hematoxylin and eosin does not always provide a clear understanding of their topography. All myoepithelial cells are located on the basal membrane and contribute to the synthesis of its components (laminin, entactin, etc.). The myoepithelial cell layer, which is consistently present in the normal mammary gland, can appear diff erently: resembling smooth muscle or epithelioid cells, having a stellate or spindle- shaped form, or not being visualized at all. It has been shown that p63 is a sensitive and specifi c nuclear myoepithelial marker, expressed in myoepithelial cells of the normal mammary gland, in residual lobular acini, sclerosing adenosis, carcinoma in situ, and not detected in invasive carcinomas. It can be included in immunohistochemical panels aimed at identifying myoepithelial cells in diagnostically challenging breast pathologies.Conclusions. Myoepithelial cells in the mammary glands play a key role in their function, facilitating the process of milk secretion. However, in certain cases, they also play an important role in the development of neoplastic transformations. Intercellular connections between secretory and myoepithelial cells indicate that the last ones suppress both the progression of carcinoma in situ to invasive breast cancer and the carcinomainduced angiogenesis. The use of the immunohistochemical marker p63 is mandatory in the panel of antibodies to identify myoepithelial cells in the mammary gland and diff erentiate between benign and malignant lesions.

Isogenic Mammary Models of Intraductal Carcinoma Reveal Progression to Invasiveness in the Absence of a Non-Obligatory In Situ Stage.

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

Prediction of Lymphovascular and Perineural Invasion of Oral Squamous Cell Carcinoma by Combined Expression of p63 and Cyclin D1.

The aim of this study was to determine the value of immune expression of p63 and cyclin D1 in the prediction of lymphovascular invasion (LVI) and perineural invasion (PNI) in oral squamous cell carcinoma (OSCC). Clinical and histopathologic features of 65 subjects with histologically confirmed OSCC were collected. Tissue microarray blocks representing all subjects were prepared for the immunohistochemical quantification of the nuclear expression of p63 and cyclin D1 using immune ratio plugin of image J software. Image analysis was performed by two independent pathologists. Independent samples t-test, analysis of variance, and receiver operating characteristic curve tests were used for statistical analysis. The level of significance was set at p≤ 0.05. The optimum cutoff value for the prediction of LVI for p63 and cyclin D1 was found to be 100 and 93.2, respectively, while the optimum cutoff value for the prediction of PNI for p63 and cyclin D1 was found to be 95.9 and 94, respectively. p63 and cyclin D1 expression correlated with several clinicopathologic features of the studied population. p63 expression was a significant predictor of moderate/poorly differentiated OSCC compared with well-differentiated OSCC. A parallel combination of positive p63 and cyclin D1 increased the specificity of predicting LVI from 89.1% and 67.4% for either p63 or cyclin D1, respectively, to 93.5% with a positive predictive value of 92.5%. Similarly, the parallel combination of the two markers raised the specificity of predicting PNI from 70% and 77.5% for either p63 or cyclin D1, respectively, to 90% with a positive predictive value of 86.3%. Combined overexpression of nuclear markers p63 and cyclin D1 can be considered as a valuable independent predictor of LVI and PNI, and hence tumor progression, in OSCC.

Increased CYR61 expression activates CCND1/c-Myc pathway to promote nasal epithelial cells proliferation in chronic rhinosinusitis with nasal polyps

PurposeChronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic sinonasal inflammatory disease characterized histologically by hyperplastic nasal epithelium and epithelial cells proliferation. Cysteine-rich angiogenic inducer 61 (CYR61) acts as a positive regulator of cell cycle process. Cyclin D1 (CCND1) and c-Myc play key roles in the processes of cell cycle and cell growth. The purpose of our research was to explore the expression and roles of CYR61, CCND1 and c-Myc in CRSwNP. MethodsFeaturePlot and vlnPlot functions embedded in the seurat package (version 4.1.1) of R software (version 4.2.0) were applied to explore the cellular distribution of CYR61, CCND1 and c-Myc in the single-cell RNA sequencing (scRNA-seq) dataset of nasal tissue samples. CYR61, CCND1 and c-Myc immunolabeling and mRNA levels in nasal tissue samples were assessed by immunohistochemistry and real-time PCR. Co-localization of CYR61, CCND1 and c-Myc with basal epithelial cell marker P63 was assayed using double-label immunofluorescence staining. Furthermore, we collected and cultured human nasal epithelial cells (HNEC) to assess the regulation and role of CYR61 in vitro study. ResultsCYR61, CCND1 and c-Myc were primarily expressed by nasal epithelial cells. Significant upregulation of CYR61, CCND1 and c-Myc positive cells and increased levels of CYR61, CCND1 and c-Myc mRNA were found in nasal polyps in comparison to control samples. Of note, CYR61 mRNA and protein levels were altered by SEB, LPS, IFN-γ, IL-13, IL-17A and TGF-β1 in HNEC. In addition, CYR61 intervention could increase CCND1 and c-Myc mRNA and protein levels to promote HNEC proliferation, and siRNA against ITGA2 (si-ITGA2) could reverse CYR61 induced upregulation of CCND1 and c-Myc mRNA and protein levels in HNEC and cell proliferation of HNEC. ConclusionsCYR61, CCND1 and c-Myc were primarily expressed by epithelial cells in nasal mucosa. CYR61, CCND1 and c-Myc expression levels were increased in CRSwNP compared with controls. CYR61 could interact with ITGA2 to enhance HNEC proliferation via upregulating CCND1 and c-Myc levels in the HNEC, leading to hyperplastic nasal epithelium in CRSwNP.