Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

KMT2D regulates p63 target enhancers to coordinate epithelial homeostasis.

Epithelial tissues rely on a highly coordinated balance between self-renewal, proliferation, and differentiation, disruption of which may drive carcinogenesis. The epigenetic regulator KMT2D (MLL4) is one of the most frequently mutated genes in all cancers, particularly epithelial cancers, yet its normal function in these tissues is unknown. Here, we identify a novel role for KMT2D in coordinating this fine balance, as depletion of KMT2D from undifferentiated epidermal keratinocytes results in reduced proliferation, premature spurious activation of terminal differentiation genes, and disorganized epidermal stratification. Genome-wide, KMT2D interacts with p63 and is enriched at its target enhancers. Depletion of KMT2D results in a broad loss of enhancer histone modifications H3 Lys 4 (H3K4) monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac) as well as reduced expression of p63 target genes, including key genes involved in epithelial development and adhesion. Together, these results reveal a critical role for KMT2D in the control of epithelial enhancers and p63 target gene expression, including the requirement of KMT2D for the maintenance of epithelial progenitor gene expression and the coordination of proper terminal differentiation.

ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes.

SignificanceΔNp63 is a master regulator of skin homeostasis since it finely controls keratinocyte differentiation and proliferation. Here, we provide cellular and molecular evidence demonstrating the functional role of a ΔNp63 interactor, the R-loop-resolving enzyme Senataxin (SETX), in fine-tuning keratinocyte differentiation. We found that SETX physically binds the p63 DNA-binding motif present in two early epidermal differentiation genes, Keratin 1 (KRT1) and ZNF750, facilitating R-loop removal over their 3' ends and thus allowing efficient transcriptional termination and gene expression. These molecular events translate into the inability of SETX-depleted keratinocytes to undergo the correct epidermal differentiation program. Remarkably, SETX is dysregulated in cutaneous squamous cell carcinoma, suggesting its potential involvement in the pathogenesis of skin disorders.

Characterization of p53/p63/p73 and Myc expressions during embryogenesis of the sea urchin.

BACKGROUND: Some marine invertebrate organisms are considered not to develop tumors due to unknown mechanisms. To gain an initial insight into how tumor-related genes may be expressed and function during marine invertebrate development, we here leverage sea urchin embryos as a model system and characterize the expressions of Myc and p53/p63/p73 which are reported to function synergistically in mammalian models as an oncogene and tumor suppressor, respectively. RESULTS: During sea urchin embryogenesis, a combo gene of p53/p63/p73 is found to be maternally loaded and decrease after fertilization both in transcript and protein, while Myc transcript and protein are zygotically expressed. p53/p63/p73 and Myc proteins are observed in the cytoplasm and nucleus of every blastomere, respectively, throughout embryogenesis. Both p53/p63/p73 and Myc overexpression results in compromised development with increased DNA damage after the blastula stage. p53/p63/p73 increases the expression of parp1, a DNA repair/cell death marker gene, and suppresses endomesoderm gene expressions. In contrast, Myc does not alter the expression of specification genes or oncogenes yet induces disorganized morphology. CONCLUSIONS: p53/p63/p73 appears to be important for controlling cell differentiation, while Myc induces disorganized morphology yet not through conventional oncogene regulations or apoptotic pathways during embryogenesis of the sea urchin.

Following the p63/Keratin5 basal cells in the sensory and non-sensory epithelia of the vomeronasal organ.

The vomeronasal organ (VNO) is a part of the accessory olfactory system, which detects pheromones and chemical factors that trigger a spectrum of sexual and social behaviors. The vomeronasal epithelium (VNE) shares several features with the epithelium of the main olfactory epithelium (MOE). However, it is a distinct neuroepithelium populated by chemosensory neurons that differ from the olfactory sensory neurons in cellular structure, receptor expression, and connectivity. The vomeronasal organ of rodents comprises a sensory epithelium (SE) and a thin non-sensory epithelium (NSE) that morphologically resembles the respiratory epithelium. Sox2-positive cells have been previously identified as the stem cell population that gives rise to neuronal progenitors in MOE and VNE. In addition, the MOE also comprises p63 positive horizontal basal cells, a second pool of quiescent stem cells that become active in response to injury. Immunolabeling against the transcription factor p63, Keratin-5 (Krt5), Krt14, NrCAM, and Krt5Cre tracing experiments highlighted the existence of horizontal basal cells distributed along the basal lamina of SE of the VNO. Single cell sequencing and genetic lineage tracing suggest that the vomeronasal horizontal basal cells arise from basal progenitors at the boundary between the SE and NSE proximal to the marginal zones. Moreover, our experiments revealed that the NSE of rodents is, like the respiratory epithelium, a stratified epithelium where the p63/Krt5+ basal progenitor cells self-replicate and give rise to the apical columnar cells facing the lumen of the VNO.

Prognostic Value of P63 Expression in Muscle-Invasive Bladder Cancer and Association with Molecular Subtypes-Preliminary Report.

There is an ongoing need for biomarkers that could reliably predict the outcome of BC and that could guide the management of this disease. In this setting, we aimed to explore the prognostic value of the transcription factor P63 in patients with muscle-invasive bladder cancer (MIBC) having undergone radical cystectomy. The correlation between P63 expression and clinicopathological features (tumor stage, nodes involvement, patterns of muscularis propria invasion, papillary architecture, anaplasia, concomitant carcinoma in situ, lymphovascular invasion, perineural invasion, necrosis) and molecular subtyping (basal and luminal type tumors) was tested in 65 radical cystectomy specimens and matched with cancer-specific survival (CSS) and overall survival (OS). P63-negative tumors displayed significantly higher rates of pattern 2 of muscularis propria invasion (50% vs. 14%, p = 0.002) and variant histology (45% vs. 19%, p = 0.022) compared to P63-positive ones. According to the combined expression of CK5/6 and CK20 (Algorithm #1), P63-positive and P63-negative tumors were mostly basal-like and double-negative, respectively (p = 0.004). Using Algorithm #2, based on the combined expression of CK5/6 and GATA3, the vast majority of tumors were luminal overall and in each group (p = 0.003). There was no significant difference in CSS and OS between P63-positive and P63-negative tumors, but the former featured a trend towards longer OS. Though associated with pathological features harboring negative prognostic potential, P63 status as such failed to predict CSS and OS. That said, it may contribute to better molecular subtyping of MIBC.

Targeted DNA sequencing in diagnosis of malignant phyllodes tumors with emphasis on tumors with keratin and p63 expression.

The differential diagnosis of malignant spindle cell neoplasms in the breast most frequently rests between malignant phyllodes tumor (MPT) and metaplastic carcinoma (MBC). Diagnosis of MPT can be challenging due to diffuse stromal overgrowth, keratin (CK) and/or p63 immunopositivity, and absent CD34 expression, which can mimic MBC, especially in core biopsies. Distinction of MPT from MBC has clinical implications, with differences in surgical approach, chemotherapy, and radiation. In this study, we evaluated MPT (78 tumors, 64 patients) for stromal CK, p63, and CD34 expression and profiled a subset (n=31) by targeted next-generation DNA sequencing (NGS), with comparison to MBC (n=44). Most MPT (71%) were CK+ and/or p63+, including 32% CK+ (25/77 focal) and 65% p63+ (32/66 focal, 10/66 patchy, 1/66 diffuse). Thirty-percent of MPT expressed both CK and p63 (20/66), compared to 95% of MBC (40/42, p<0.001). CK and/or p63 were positive in CD34+ and CD34- MPT. Recurrent genetic aberrations in MPT involved TERT, TP53, MED12, CDKN2A, chromatin modifiers, growth factor receptors/ligands, and PI-3K and MAPK pathway genes. Only MED12 (39%, 12/31) and SETD2 (13%, 4/31) were exclusively mutated in MPT and not MBC (p<0.001 and p=0.044, respectively), whereas PIK3R1 mutations were only found in MBC (35%, 13/35, p<0.001). Comparative literature review additionally identified ARID1B, EGFR, FLNA, NRAS, PDGFRB, RAD50, and RARA alterations enriched or exclusively in MPT versus MBC. MED12 was mutated in MPT with diffuse stromal overgrowth (53%, 9/17), CD34- MPT (41%, 7/17), and CK+ and/or p63+ MPT (39%, 9/23), including 36% of CD34- MPT with CK and/or p63 expression. Overall, MED12 mutation and/or CD34 expression were observed in 68% (21/31) MPT, including 61% (14/23) of CK+ and/or p63+ tumors. Our results emphasize the prevalence of CK and p63 expression in MPT and demonstrate diagnostic utility of NGS, especially in MPT with confounding factors that can mimic MBC.

Tumour 63 protein (p63) in breast pathology: biology, immunohistochemistry, diagnostic applications, and pitfalls.

Tumour protein 63 (p63) is a transcription factor of the p53 gene family, encoded by the TP63 gene located at chromosome 3q28, which regulates the activity of genes involved in growth and development of the ectoderm and derived tissues. p63 protein is normally expressed in the nuclei of the basal cell layer of glandular organs, including breast, in squamous epithelium and in urothelium. p63 immunohistochemical (IHC) staining has several applications in diagnostic breast pathology. It is commonly used to demonstrate myoepithelial cells at the epithelial stromal interface to differentiate benign and in situ lesions from invasive carcinoma and to characterize and classify papillary lesions including the distinction of breast intraduct papilloma from skin hidradenoma. p63 IHC is also used to identify and profile lesions showing myoepithelial cell and/or squamous differentiation, e.g. adenomyoepithelioma, salivary gland-like tumours including adenoid cystic carcinoma, and metaplastic breast carcinoma including low-grade adenosquamous carcinoma. This article reviews the applications of p63 IHC in diagnostic breast pathology and outlines a practical approach to the diagnosis and characterization of breast lesions through the identification of normal and abnormal p63 protein expression. The biology of p63, the range of available antibodies with emphasis on staining specificity and sensitivity, and pitfalls in interpretation are also discussed. The TP63 gene in humans, which shows a specific genomic structure, resulting in either TAp63 (p63) isoform or ΔNp63 (p40) isoform. As illustrated in the figure, both isoforms contain a DNA-binding domain (Orange box) and an oligomerization domain (Grey box). TAp63 contains an N-terminal transactivation (TA) domain (Green box), while ΔNp63 has an alternative terminus (Yellow box). Antibodies against conventional pan-p63 (TP63) bind to the DNA binding domain common to both isoforms (TAp63 and p40) and does not distinguish between them. Antibodies against TAp63 bind to the N-terminal TA domain, while antibodies specific to ΔNp63 (p40) bind to the alternative terminus. Each isoform has variant isotypes (α, ß, γ, δ, and ε).

ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFß-induced cancer cell stemness and invasiveness.

BACKGROUND: p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor ß (TGFß) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFß activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFß and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported. METHODS: Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription. RESULTS: The sphere-forming capacity of breast cancer cells was enhanced upon TGFß stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFß signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFß stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFß effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFß as the upstream signal for guiding ΔNp63 to the TGFß/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFß through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription. CONCLUSIONS: ΔNp63, phosphorylated and recruited by TGFß to the TGFß/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

ΔNp63 overexpression promotes oral cancer cell migration through hyperactivated Activin A signaling.

Oral cancer is a common malignant tumor of the oral cavity that affects many countries with a prevalent distribution in the Indian subcontinent, with poor prognosis rate on account of locoregional metastases. Gain-of-function mutations in p53 and overexpression of its related transcription factor, p63 are both widely reported events in oral cancers. However, targeting these alterations remains a far-achieved aim due to lack of knowledge on their downstream signaling pathways. In the present study, we characterize the isoforms of p63 and using knockdown strategy, decipher the functions and oncogenic signaling of p63 in oral cancers. Using Microarray and Chromatin Immunoprecipitation experiments, we decipher a novel transcriptional regulatory axis between p63 and Activin A and establish its functional significance in migration of oral cancer cells. Using an orally bioavailable inhibitor of the Activin A pathway to attenuate oral cancer cell migration and invasion, we further demonstrate the targetability of this signaling axis. Our study highlights the oncogenic role of ΔNp63 - Activin A - SMAD2/3 signaling and provides a basis for targeting this oncogenic pathway in oral cancers.

Mammalian esophageal stratified tissue homeostasis is maintained distinctively by the epithelial pluripotent p63+Sox2+ and p63-Sox2+ cell populations.

Self-renewing, damage-repair and differentiation of mammalian stratified squamous epithelia are subject to tissue homeostasis, but the regulation mechanisms remain elusive. Here, we investigate the esophageal squamous epithelial tissue homeostasis in vitro and in vivo. We establish a rat esophageal organoid (rEO) in vitro system and show that the landscapes of rEO formation, development and maturation trajectories can mimic those of rat esophageal epithelia in vivo. Single-cell RNA sequencing (scRNA-seq), snapshot immunostaining and functional analyses of stratified "matured" rEOs define that the epithelial pluripotent stem cell determinants, p63 and Sox2, play crucial but distinctive roles for regulating mammalian esophageal tissue homeostasis. We identify two cell populations, p63+Sox2+ and p63-Sox2+, of which the p63+Sox2+ population presented at the basal layer is the cells of origin required for esophageal epithelial stemness maintenance and proliferation, whereas the p63-Sox2+ population presented at the suprabasal layers is the cells of origin having a dual role for esophageal epithelial differentiation (differentiation-prone fate) and rapid tissue damage-repair responses (proliferation-prone fate). Given the fact that p63 and Sox2 are developmental lineage oncogenes and commonly overexpressed in ESCC tissues, p63-Sox2+ population could not be detected in organoids formed by esophageal squamous cell carcinoma (ESCC) cell lines. Taken together, these findings reveal that the tissue homeostasis is maintained distinctively by p63 and/or Sox2-dependent cell lineage populations required for the tissue renewing, damage-repair and protection of carcinogenesis in mammalian esophagi.

How MicroRNAs Command the Battle against Cancer.

MicroRNAs (miRNAs) are small RNA molecules that regulate more than 30% of genes in humans. Recent studies have revealed that miRNAs play a crucial role in tumorigenesis. Large sets of miRNAs in human tumors are under-expressed compared to normal tissues. Furthermore, experiments have shown that interference with miRNA processing enhances tumorigenesis. Multiple studies have documented the causal role of miRNAs in cancer, and miRNA-based anticancer therapies are currently being developed. This review primarily focuses on two key points: (1) miRNAs and their role in human cancer and (2) the regulation of tumor suppressors by miRNAs. The review discusses (a) the regulation of the tumor suppressor p53 by miRNA, (b) the critical role of the miR-144/451 cluster in regulating the Itch-p63-Ago2 pathway, and (c) the regulation of PTEN by miRNAs. Future research and the perspectives of miRNA in cancer are also discussed. Understanding these pathways will open avenues for therapeutic interventions targeting miRNA regulation.

ABCC1 Is a ΔNp63 Target Gene Overexpressed in Squamous Cell Carcinoma.

The transcription factor ΔNp63 plays a pivotal role in maintaining the integrity of stratified epithelial tissues by regulating the expression of distinct target genes involved in lineage specification, cell stemness, cell proliferation and differentiation. Here, we identified the ABC transporter subfamily member ABCC1 as a novel ΔNp63 target gene. We found that in immortalized human keratinocytes and in squamous cell carcinoma (SCC) cells, ∆Np63 induces the expression of ABCC1 by physically occupying a p63-binding site (p63 BS) located in the first intron of the ABCC1 gene locus. In cutaneous SCC and during the activation of the keratinocyte differentiation program, ∆Np63 and ABCC1 levels are positively correlated raising the possibility that ABCC1 might be involved in the regulation of the proliferative/differentiative capabilities of squamous tissue. However, we did not find any gross alteration in the structure and morphology of the epidermis in humanized hABCC1 knock-out mice. Conversely, we found that the genetic ablation of ABCC1 led to a marked reduction in inflammation-mediated proliferation of keratinocytes, suggesting that ABCC1 might be involved in the regulation of keratinocyte proliferation upon inflammatory/proliferative signals. In line with these observations, we found a significant increase in ABCC1 expression in squamous cell carcinomas (SCCs), a tumor type characterized by keratinocyte hyper-proliferation and a pro-inflammatory tumor microenvironment. Collectively, these data uncover ABCC1 as an additional ∆Np63 target gene potentially involved in those skin diseases characterized by dysregulation of proliferation/differentiation balance.

TAF15::NR4A3 gene fusion identifies a morphologically distinct subset of extraskeletal myxoid chondrosarcoma mimicking myoepithelial tumors.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain differentiation predominantly arising in deep soft tissue. Its conventional morphologic appearance manifests as a relatively well-circumscribed, multilobular tumor composed of uniform short spindle-to-ovoid primitive mesenchymal cells with deeply eosinophilic cytoplasm arranged in anastomosing cords within abundant myxoid matrix. The genetic hallmark of EMC has long been considered to be pathognomonic gene rearrangements involving NR4A3, which when fused to TAF15, often have high-grade morphology with increased cellularity, moderate to severe cytologic atypia, and rhabdoid cytomorphology. Herein, we describe two cases of EMC with TAF15::NR4A3 fusion that appear morphologically distinct from both conventional and high-grade EMC. Both cases had an unusual biphasic appearance and showed diffuse positivity for p63, mimicking myoepithelial tumors. DNA methylation profiling demonstrated that both cases clearly cluster with EMC, indicating that they most likely represent morphologically distinct variants of EMC. The clinical significance and prognostic impact of this morphologic variance remains to be determined. Molecular testing, including DNA methylation profiling, can help to confirm the diagnosis and avoid confusion with mimics; it adds another layer of data to support expanding the morphologic spectrum of EMC.

The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett's Neoplasia.

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

Mucoepidermoid carcinoma may be devoid of squamoid cells by immunohistochemistry: expanding the histologic and immunohistochemical spectrum of MAML2- rearranged salivary gland tumours.

Mucoepidermoid carcinoma (MEC) is historically defined by a mix of squamoid, intermediate, and mucous cells, but we have recently encountered several cases lacking immunoreactivity for squamous markers p40, p63, and CK5/6 despite MAML2 fusions. This study will characterise these unique tumours. Ten MEC were collected arising from the parotid gland (n = 4), submandibular gland (n = 2), nasopharynx (n = 1), base of tongue (n = 1), bronchus (n = 1), and trachea (n = 1). Six tumours were low-grade, two intermediate-grade, one high-grade, and one demonstrated low-grade areas with high-grade transformation. Four cases were oncocytic, four had clear-cell features, two had spindle cell features, and one high-grade MEC had prominent solid, cord-like, and micropapillary features. The tumours were negative for p40 (10/10), p63 (10/10), and CK5/6 (9/9). Targeted RNA sequencing demonstrated CRTC1::MAML2 in five cases, CRTC3::MAML2 in two, and a novel MAML2::CEP126 in the unusual high-grade case. In two cases with insufficient RNA, MAML2 fluorescence in situ hybridisation (FISH) showed rearrangement. Genetically-confirmed MEC may lack overt squamous differentiation by histology and immunohistochemistry. While most cases harboured canonical fusions and fit within the spectra of MEC variants with oncocytic, clear cell, and/or spindle cell features, one had a novel MAML2::CEP126 fusion and unusual morphology. In MEC without squamoid cells, the use of immunohistochemistry may hinder, rather than aid, the correct diagnosis. In such cases, MAML2 analysis is most useful. The historical definition of MEC as a carcinoma with squamoid, intermediate and mucous cells should be revisited.

Low-grade adenosquamous carcinoma of the breast: a clinical, morphological and immunohistochemical analysis of 25 patients.

AIMS: Due to its rarity and non-specific clinical and pathological features, low-grade adenosquamous carcinoma (LGASC) of the breast continues to pose diagnostic challenges. Unlike other triple-negative breast carcinomas, LGASC tends to have an indolent clinical behaviour. It is essential to recognise this lesion for accurate diagnosis and appropriate management. METHODS AND RESULTS: Twenty-five cases of LGASC were identified in our archives and collaborating institutes. Cases of LGASC with dominant coexisting other type carcinomas were excluded. We studied the clinical presentation, morphological features, patterns of the commonly used immunohistochemical stains and follow-up. In our cohort, LGASC was commonly located at the outer aspect of the breast and associated with intraductal papilloma. The morphology of LGASC is characterised by infiltrating small glands and nests with variable squamous differentiation. We also found cuffing desmoplastic (fibrolamellar) stromal change in 75% of patients and peripheral lymphocytic aggregates in 87.5% of patients. P63 and smooth muscle myosin (SMM) were the most common myoepithelial markers used to assist in diagnosis. P63 often stained peripheral tumour cells surrounding invasive glands (circumferential staining in 80% of the cases), mimicking myoepithelial cells. It also stained the small nests with squamous differentiation. However, SMM was negative in 63% of the cases. The vast majority of our cases were triple-negative; only a few had focal and weak expressions of ER and PR. One patient who did not have excision developed lymph node metastasis. Most patients underwent excision or mastectomy with negative margins as surgical treatment; there were no recurrences or metastases in these patients with clinical follow-ups up to 108 months. CONCLUSIONS: LGASC has some unique, although not entirely specific, morphological features and immunohistochemical staining patterns. Fibrolamellar stromal change, peripheral lymphocytic aggregates and variable staining of p63 and SMM are valuable features to facilitate the diagnosis.

The role of p63 in embryonic external genitalia outgrowth in mice.

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh-Wnt/Ctnnb1-Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.

NONO::TFE3 fusion cutaneous epithelioid and spindle cell tumor: A case series.

The NONO::TFE3 fusion has been described in MiT family translocation renal cell carcinomas as well as extracutaneous perivascular epithelioid cell tumors (PEComas). PEComas are known to express myogenic and melanocytic markers but SOX10 and p63 positivity has never been reported. We report two primary cutaneous tumors that morphologically and molecularly fit PEComas, both harboring the NONO::TFE3 fusion, but with an unusual immunophenotype of SOX10 and p63 positivity. One case was on an 80-year-old male's finger, and the other one was on a 72-year-old female's thigh. Both were well-circumscribed multinodular dermal tumors composed of nests of monotonous epithelioid to spindled cells with pale to vacuolated cytoplasm, some of which were arranged around blood vessels. Both tumors were positive for SOX10, S100, and p63, focally positive for Melan-A, and negative for myogenic markers. There are very little data regarding the molecular findings of primary cutaneous PEComas. While the NONO::TFE3 fusion has been identified in extracutaneous PEComas, it has never been reported in primary cutaneous cases. We believe these cases represent a previously undescribed subtype of cutaneous tumor which shows some immunophenotypic expression of melanocytic markers and we named these cases NONO::TFE3 fusion cutaneous epithelioid and spindle cell tumor. Further, we raise the question of whether this tumor should fall under the rubric of PEComa because of its morphology, partial expression of melanocytic markers, and the presence of the NONO::TFE3 fusion, or whether these tumors represent a separate novel class of tumors since the immunophenotypic expression of SOX10 and p63 is unusual for PEComas.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.