Basal Markers Research Articles

To study the expression of cytokeratin 5 and 6 in triple negative breast carcinoma: An immunohistochemical study

Introduction: Breast cancer is the second most common cancer developing in women worldwide Triple Negative Breast Carcinomas (TNBC) are regarded as one of the most malignant phenotypes, Immunohisto-chemically these tumors are endocrine receptor negative, HER2 –receptor negative, with expression of basal markers - Cytokeratin 5 /6, carrying a poor prognosis. Effective evaluation of these tumor markers helps in efficient treatment of the patient with all these markers carrying a prognostic significance. The current study helps in identifying Cytokeratin 5/6 as a potential multifaceted biomarker and in identifying novel therapeutic agents for Triple Negative Breast Carcinomas.Materials and Methods: The present immunohistochemical study was conducted on 40 histologically proven cases of triple negative breast carcinoma (TNBC) which were further subjected to Cytokeratin 5/6 expression to determine prognostic and therapeutic value.Result: Cytokeratin 5/6 expression was seen in 16 cases of TNBC, out of 40 histologically proven cases of TNBC. The maximum number of patients of triple negative breast carcinoma were in the age group of 41-50 years comprising 40% of the total cases. % of the total cases. Maximum number of cases of triple negative breast carcinoma were of grade III, constituting 60% followed by grade II Conclusion: According to the current study, Cytokeratin 5/6 positivity has got direct relationship with the grade of the tumour, lymph nodal status and lymphovascular invasion in TNBC cases. TNBC have a grave clinical outcome and decreased 5 year survival rate. Thus, it is highlighted that all TNBC patients should be stained by CK 5/6 and if found positive for CK 5/6 expression it, implies basal like phenotype and aggressive intervention is required. Cytokeratin 5/6 carries a diagnostic as wells as therapeutic importance.

AtlasGrabber: a software facilitating the high throughput analysis of the human protein atlas online database

BackgroundThe human protein atlas (HPA) is an online database containing large sets of protein expression data in normal and cancerous tissues in image form from immunohistochemically (IHC) stained tissue microarrays. In these, the tissue architecture is preserved and thus provides information on the spatial distribution and localization of protein expression at the cellular and extracellular levels. The database is freely available online through the HPA website but currently without support for large-scale screening and analysis of the images in the database. Features like spatial information are typically lacking in gene expression datasets from homogenized tissues or single-cell analysis. To enable high throughput analysis of the HPA database, we developed the AtlasGrabber software. It is available freely under an open-source license. Based on a predefined gene list, the software fetches the images from the database and displays them for the user. Several filters for specific antibodies or images enable the user to customize her/his image analysis. Up to four images can be displayed simultaneously, which allows for the comparison of protein expression between different tissues and between normal and cancerous tissues. An additional feature is the XML parser that allows the extraction of a list of available antibodies, images, and genes for specific tissues or cancer types from the HPA’s database file.ResultsCompared to existing software designed for a similar purpose, ours provide more functionality and is easier to use. To demonstrate the software’s usability, we identified six new markers of basal cells of the prostate. A comparison to prostate cancer showed that five of them are absent in prostate cancer.ConclusionsThe HPA is a uniquely valuable database. By facilitating its usefulness with the AtlasGrabber, we enable researchers to exploit its full capacity. The loss of basal cell markers is diagnostic for prostate cancer and can help refine the histopathological diagnosis of prostate cancer. As proof of concept, with the AtlasGrabber we identified five new potential biomarkers specific for prostate basal cells which are lost in prostate cancer and thus can be used for prostate cancer diagnostics.

Nonmalignant AR-positive prostate epithelial cells and cancer cells respond differently to androgen.

Prostate cancer research suffers from the lack of suitable models to study the role of normal cells in prostate carcinogenesis. To address this challenge, we developed a cell line model mimicking luminal prostate epithelial cells by modifying the immortalized prostate epithelial cell line RWPE-1 to constitutively express the androgen receptor (AR). RWPE-1-AR cells express known AR target genes, and exhibit coexpression of luminal and basal markers characteristic of transient amplifying cells, and an RNA signature resembling prostate luminal progenitor cells. Under unstimulated conditions, constitutive AR expression does not have a biologically significant effect on the proliferation of RWPE-1 cells, but when stimulated by androgens, growth is retarded. The transcriptional response of RWPE-1-AR cells to androgen stimulation involves suppression of the growth-related KRAS pathway and is thus markedly different from that of the prostate cancer cell line LNCaP and its derivative AR-overexpressing LNCaP-ARhi cells, in which growth- and cancer-related pathways are upregulated. Hence, the nonmalignant AR-positive RWPE-1-AR cell line model could be used to study the transformation of the prostate epithelium.

Abstract A031: Proteogenomic analysis of the PDAC epithelium reveals tissue markers of subtype identity and biology

Abstract Effective treatments for Pancreatic ductal adenocarcinoma (PDAC) remain an urgent need. Integration of molecular PDAC subtypes into clinical trials could enable translational insights into how we might refine existing and emerging therapies to improve treatment options, yet this requires robust clinically suitable marker genes and a better understanding of subtype-related biology. Here, we deeply profiled the molecular composition of human PDAC epithelia and assessed differentially expressed genes for their ability to discriminate between subtypes and mirror their key biological traits. Using well-annotated resected & advanced biospecimens, we mapped the stromal and epithelial landscapes of PDAC subtypes by combining compartment-specific transcriptomics and proteomics with quantitative image analysis of a 42-marker IHC panel and single cell RNA sequencing analyses. The integrated profiling yielded a shortlist of 30 genes that were differentially expressed at both transcriptomic and proteomic levels, and across primary and metastatic sites. These were then scrutinized for their suitability as potential single gene subtype markers through association with subtype-related epithelial and stromal biology and clinical outcomes, and via practical considerations such as immunohistochemical staining quality and cell type-specific expression patterns. Since PDAC subtypes frequently co-occur intratumorally, we furthermore assessed complementarity of different subtype marker combinations and tested association of individual markers with regional subtype biology. Annexin A8 emerged as strong IHC-suitable marker gene of the basal/squamous subtype, that exhibited prognostic and predictive value. Concordantly, Annexin A8 expression was highly correlated with TP63, a master transcription factor for the squamous subtype, and CK5, a well-established basal cell marker. In tissue stains and single cell RNAseq data, Annexin A8 specifically marked malignant epithelia but was absent from adjacent normal acini and stroma cell populations. Annexin A8 expression in PDAC epithelia was spatially distinct from the known classical subtype biomarker GATA6, as well as Claudin 18, which was the top classical/pancreatic progenitor marker gene in our integrated multiOMIC analysis. Furthermore, Annexin A8high tumors as well as Annexin A8high intratumoral regions both recapitulated key biological traits of the basal-like/squamous PDAC subtype, such as increased EMT marker expression, proliferation, hypoxia, macrophage attraction and T cell repulsion. In conclusion, integrated proteogenomic characterization of PDAC subtypes and their tissue features uncovers complementary subtype marker pairs with clinical potential and provides insights into subtype-specific biology with implications in the development of future therapeutic approaches tailored to each PDAC subtype. Citation Format: Barbara T. Grünwald, Foram Vyas, Michael Geuenich, Nathan Chan, Ricardo Gonzalez, Kazeera Aliar, Niklas Krebs, Antoine Devisme, Geoffroy Andrieux, Gun Ho Jang, Grainne O'Kane, Julie Wilson, Jennifer Knox, Faiyaz Notta, Kieran Campbell, Steven Gallinger, Melanie Boerries, Sandra Fischer, Thomas Kislinger, Rama Khokha. Proteogenomic analysis of the PDAC epithelium reveals tissue markers of subtype identity and biology [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A031.

P63-expressing Prostate Adenocarcinoma: A Case Study of a Rare Subtype

Abstract Introduction/Objective Prostate acinar adenocarcinoma (PCa) is a well-researched,and often diagnosed entity. A combination of architectural and cytologic characteristics are used in accurately identifying these carcinomas. However, the diagnosis can be challenging when the foci are minute or the cancer presents with unusual features. Ancillary studies aid in these scenarios. The loss of basal cells in adenocarcinoma makes markers targeting these cells desirable. PIN4 cocktail immunohistochemical stain, which combines alpha-methylacyl-coA racemase (AMACR), that is overexpressed in prostate carcinoma, with basal markers high molecular weight cytokeratin and p63, is a popular ancillary study to assist diagnosing demanding cases. Herein, we present a rare, challenging subtype of prostate carcinoma expressing p63. Methods/Case Report A 66-year-old male, with a past medical history of treated HIV and hyperlipidemia, underwent screening biopsies for a PSA of 7.5 ng/mL. Out of 22 parts, three cores showed modest amounts of atypical infiltrating cells. The architecture comprised of ill-defined glands, individual cells and single files of cells. They possessed high nucleocytoplasmic ratio giving a hint of atrophy, with occasional spindling and prominent nucleoli. To add to the challenge, PIN4 immunostaining showed strong nuclear expression of a basal marker with negative AMACR. Due to unusual morphology and basal marker expression, a consideration of basal cell lesion was entertained. However, subsequent staining revealed negative expression of HMWCK, and positive expressions of NKX3.1 and p40. Overall, with this morphology and immunoprofile we diagnosed our case as prostate carcinoma with aberrant expression of p63. Results (if a Case Study enter NA) N/A Conclusion Grading these lesions is controversial as they are considered to carry a better prognosis over conventional acinar adenocarcinoma of similar grade. A distinct etiopathology is suspected as molecular tests for ERG and PTEN are without alterations. Further discussion and characterization of these lesions is warranted as they are infrequently encountered and prone to being overlooked due to their similarities to benign basal cells.

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

Arl13b controls basal cell stemness properties and Hedgehog signaling in the mouse epididymis.

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

Chronic Arsenic Exposure Upregulates the Expression of Basal Transcriptional Factors and Increases Invasiveness of the Non-Muscle Invasive Papillary Bladder Cancer Line RT4.

The bladder is a target organ for inorganic arsenic, a carcinogen and common environmental contaminant found in soil and water. Urothelial carcinoma (UC) is the most common type of bladder cancer (BC) that develops into papillary or non-papillary tumors. Papillary tumors are mostly non-muscle invasive (NMIUC), easier treated, and have a better prognosis. Urothelial carcinoma can be molecularly sub-typed as luminal or basal, with papillary tumors generally falling into the luminal category and basal tumors exclusively forming muscle invasive urothelial carcinomas (MIUC). It is unclear why some UCs develop more aggressive basal phenotypes. We hypothesized that chronic arsenic exposure of a papillary luminal bladder cancer would lead to the development of basal characteristics and increase in invasiveness. We treated the human papillary bladder cancer cell line RT4 with 1 µM arsenite (As3+) for twenty passages. Throughout the study, key luminal and basal gene/protein markers in the exposed cells were evaluated and at passage twenty, the cells were injected into athymic mice to evaluate tumor histology and measure protein markers using immunohistochemistry. Our data indicates that chronic As3+- treatment altered cellular morphology and decreased several luminal markers in cell culture. The histology of the tumors generated from the As3+-exposed cells was similar to the parent (non-treated) however, they appeared to be more invasive in the liver and displayed elevated levels of some basal markers. Our study demonstrates that chronic As3+ exposure is able to convert a non-invasive papillary bladder cancer to an invasive form that acquires some basal characteristics.

The interconnected relationships between middle ear bulla size, cavitation defects, and chronic otitis media revealed in a syndromic mouse model.

High incidence of chronic otitis media is associated with human craniofacial syndromes, suggesting that defects in the formation of the middle ear and associated structures can have a knock-on effect on the susceptibility to middle ear inflammation. Patients with branchio-oto-renal (BOR) syndrome have several defects in the ear leading to both sensorineural and conductive hearing loss, including otitis media. 40% of BOR syndrome cases are due to Eya1 haploinsufficiency, with mouse models affecting Eya1, mimicking many of the defects found in patients. Here, we characterize the onset, consequences, and underlying causes of chronic otitis media in Eya1 heterozygous mice. Cavitation defects were evident in these mice from postnatal day (P)11 onwards, with mesenchyme around the promontory and attic regions of the middle ear space. This mesenchyme was still prominent in adult Eya1 heterozygous mice, while the wild-type littermates had fully aerated ears from P14 onwards. MicroCT analysis highlighted a significantly smaller bulla, confirming the link between bulla size defects and the ability of the mesenchyme to retract successfully. Otitis media was observed from P14, often presenting unilaterally, resulting in hyperplasia of the middle ear mucosa, expansion of secretory cells, defects in the motile cilia, and changes in basal epithelial cell markers. A high incidence of otitis media was identified in older mice but only associated with ears with retained mesenchyme. To understand the impact of the environment, the mouse line was rederived onto a super-clean environment. Cavitation defects were still evident at early stages, but these generally resolved over time, and importantly, no signs of otitis media were observed at 6 weeks. In conclusion, we show that a small bulla size is closely linked to defects in cavitation and the presence of retained mesenchyme. A delay in retraction of the mesenchyme predates the onset of otitis media, making the ears susceptible to its development. Early exposure to OM appears to exacerbate the cavitation defect, with mesenchyme evident in the middle ear throughout the animal’s life. This highlights that permanent damage to the middle ear can arise as a consequence of the early onset of OM.

Incidence of prostatic intraepithelial neoplasia in TURP specimens with special reference to p63 expression in various lesions of prostate

Introduction: Prostate cancer is the second common malignancy in men. Prostatic intraepithelial neoplasia (PIN) is the precursor lesion of prostatic carcinoma. Histopathological examination is necessary to diagnose PIN lesions. Objective: The aim of the study is to determine the incidence of Prostatic intraepithelial neoplasia and to analyse the usefulness of basal cell marker p63 expression in various lesions of prostate. Methods: This is a two-year prospective study of 65 transurethral resection specimens of prostate, carried out in the Department of pathology, Dr B R Ambedkar Medical College, Bangalore. mmunohistochemical marker p63 is used and its expression in various lesions was analysed. Results: Out of 65 cases studied, 4 were inflammatory lesions, 52 were benign prostatic hyperplasia (BPH) and 9 were malignant lesions. Low grade PIN was identified in 8(12.3%) cases of BPH. High grade PIN was seen in 9(13.8%) cases and tufting pattern was the commonest. HGPIN was predominantly associated with adenocarcinoma. p63 was expressed in all the benign glands in BPH and HGPIN. Malignant glands were negative for p63 expression.

PPARgamma agonism inhibits progression of premalignant lesions in a murine lung squamous cell carcinoma model.

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

In Vivo Application of CRISPR/Cas9 Revealed Implication of Foxa1 and Foxp1 in Prostate Cancer Proliferation and Epithelial Plasticity.

Simple SummaryProstate cancer is diagnosed in one out of eight men, with large implications on life quality. Forkhead box proteins are often found mutated in prostate cancer but their functions are still not fully understood. In this study, we applied CRISPR to investigate the function of two Forkhead box proteins, Foxa1 and Foxp1, in the mouse prostate in combination with loss of Pten. Our results reveal that Foxp1 is a tumor suppressor in prostate cancer progression by controlling proliferation and genes regulated by the androgen receptor. Foxa1 controls cell plasticity, as loss of Foxa1 converted the prostatic luminal cells to basal cells. Hereby, this study sheds light on two distinct functions of Forkhead box proteins in prostate cancer.Prostate cancer is the most common cancer in men in the Western world and the number is rising. Prostate cancer is notoriously heterogeneous, which makes it hard to generate and study in pre-clinical models. The family of Forkhead box (FOX) transcription factors are often altered in prostate cancer with especially high mutation burden in FOXA1 and FOXP1. FOXA1 harbors loss or gain of function mutations in 8% of prostate cancer, which increases to 14% in metastatic samples. FOXP1 predominately occurs with loss of function mutations in 7% of primary tumors, and similar incidents are found in metastatic samples. Here, we applied in vivo CRISPR editing, to study the loss of functions of these two FOX transcription factors, in murine prostate in combination with loss of Pten. Deficiency of Foxp1 increased proliferation in combination with loss of Pten. In contrast, proliferation was unchanged when androgen was deprived. The expression of Tmprss2 was increased when Foxp1 was mutated in vivo, showing that Foxp1 is a repressor for this androgen-regulated target. Furthermore, analysis of FOXP1 and TMPRSS2 expression in a human prostate cancer data set revealed a negative correlation. Mutation of Foxa1 in the murine prostate induces cell plasticity to luminal cells. Here, epithelial cells with loss of Foxa1 were transdifferentiated to cells with expression of the basal markers Ck5 and p63. Interestingly, these cells were located in the lumen and did not co-express Ck8. Overall, this study reveals that loss of Foxp1 increases cell proliferation, whereas loss of Foxa1 induces epithelial plasticity in prostate cancer.

Diacetyl inhalation impairs airway epithelial repair in mice infected with influenza A virus.

Bronchiolitis obliterans (BO) is a debilitating disease of the small airways that can develop following exposure to toxic chemicals as well as respiratory tract infections. BO development is strongly associated with diacetyl (DA) inhalation exposures at occupationally relevant concentrations or severe influenza A viral (IAV) infections. However, it remains unclear whether lower dose exposures or more mild IAV infections can result in similar pathology. In the current work, we combined these two common environmental exposures, DA and IAV, to test whether shorter DA exposures followed by sublethal IAV infection would result in similar airways disease. Adult mice exposed to DA vapors 1 h/day for 5 consecutive days followed by infection with the airway-tropic IAV H3N2 (HKx31) resulted in increased mortality, increased bronchoalveolar lavage (BAL) neutrophil percentage, mixed obstruction and restriction by lung function, and subsequent airway remodeling. Exposure to DA or IAV alone failed to result in significant pathology, whereas mice exposed to DA + IAV showed increased α-smooth muscle actin (αSMA) and epithelial cells coexpressing the basal cell marker keratin 5 (KRT5) with the club cell marker SCGB1A1. To test whether DA exposure impairs epithelial repair after IAV infection, mice were infected first with IAV and then exposed to DA during airway epithelial repair. Mice exposed to IAV + DA developed similar airway remodeling with increased subepithelial αSMA and epithelial cells coexpressing KRT5 and SCGB1A1. Our findings reveal an underappreciated concept that common environmental insults while seemingly harmless by themselves can have catastrophic implications on lung function and long-term respiratory health when combined.

Multiple roles of Pax6 in postnatal cornea development

Mammalian corneal development is a multistep process, including formation of the corneal epithelium (CE), endothelium and stroma during embryogenesis, followed by postnatal stratification of the epithelial layers and continuous renewal of the epithelium to replace the outermost corneal cells. Here, we employed the Cre-loxP system to conditionally deplete Pax6 proteins in two domains of ocular cells, i.e., the ocular surface epithelium (cornea, limbus and conjunctiva) (OSE) or postnatal CE via K14-cre or Aldh3-cre, respectively. Earlier and broader inactivation of Pax6 in the OSE resulted in thickened OSE with CE and limbal cells adopting the conjunctival keratin expression pattern. More restricted depletion of Pax6 in postnatal CE resulted in an abnormal cornea marked by reduced epithelial thickness despite increased epithelial cell proliferation. Immunofluorescence studies revealed loss of intermediate filament Cytokeratin 12 and diffused expression of adherens junction components, together with reduced tight junction protein, Zonula occludens-1. Furthermore, the expression of Cytokeratin 14, a basal cell marker in apical layers, indicates impaired differentiation of CE cells. Collectively, our data demonstrate that Pax6 is essential for maintaining proper differentiation and strong intercellular adhesion in postnatal CE cells, whereas limbal Pax6 is required to prevent the outgrowth of conjunctival cells to the cornea.

Frequency and Prognostic Significance of Intertumoural Heterogeneity in Multifocal Jejunoileal Neuroendocrine Tumours.

Simple SummaryWell differentiated jejunoileal neuroendocrine tumors (SI-NETs) commonly present as multiple primaries, which are believed to be clonally unrelated. Our study aimed to explore whether these multifocal lesions show intertumoral differences between the most common histomorphological parameters and if so, whether these differences can be associated with patient prognosis. While WHO grade and standard neuroendocrine markers were found to be mostly stable, we observed intertumoral heterogeneity in the expression of SSTR2, CDX2, and serotonin, but observed no survival differences between the expression groups or in comparison to unifocal NETs. Although multifocal SI-NETs showed some degree of heterogeneity in their central morphological parameters, these findings do not appear to be of major clinical significance, rendering an extensive testing of all multifocal lesions as not necessarily required.Background: A recent study found that multifocal jejunoileal neuroendocrine tumors (SI-NETs) are genetically unrelated synchronous neoplasms. So far, it is unclear if this finding of synchronous independent neoplasms is mirrored by heterogeneity of key morphological parameters of SI-NETs and how it affects patient survival. Methods: We separately assessed WHO grade (based on the Ki-67 index), expression of basal diagnostic markers (synaptophysin/chromogranin A/CDX2/serotonin), SSTR2a, and the contexture of the immunogenic microenvironment in 146 separate tumors from 28 patients with multifocal SI-NETs and correlated the results with clinicopathological factors and survival. Results: Synaptophysin and chromogranin A were strongly expressed in all tumors. WHO grade was concordant within all multifocal lesions in more than 80% of cases and the highest grade was usually found in the most advanced primary. Intertumoral expression of serotonin, SSTR2, and CDX2 was discrepant in 32%, 43%, and 50% of all patients, respectively. Neither heterogeneity of any of the aforementioned markers nor multifocality itself had any impact on patient survival (p = n.s.). Discussion: Multifocal SI-NET show considerable variability in some of the central diagnostic parameters. However, neither intertumoral heterogeneity of those parameters nor multifocality itself had any impact on patient survival, showing that extensive testing of all multifocal lesions is not necessarily required.

Human Umbilical Cord Lining-Derived Epithelial Cells: A Potential Source of Non-Native Epithelial Cells That Accelerate Healing in a Porcine Cutaneous Wound Model.

Human umbilical cord lining epithelial cells [CLECs) are naïve in nature and can be ethically recovered from cords that are routinely discarded. The success of using oral mucosal epithelial cells for cornea defects hints at the feasibility of treating cutaneous wounds using non-native CLECs. Herein, we characterized CLECs using flow cytometry (FC) and skin organotypic cultures in direct comparison with skin keratinocytes (KCs). This was followed by wound healing study to compare the effects of CLEC application and the traditional use of human skin allografts (HSGs) in a porcine wound model. While CLECs were found to express all the epidermal cell markers probed, the major difference between CLECs and KCs lies in the level of expression (in FC analysis) as well as in the location of expression (of the epithelium in organotypic cultures) of some of the basal cell markers probed. On the pig wounds, CLEC application promoted accelerated healing with no adverse reaction compared to HSG use. Though CLECs, like HSGs, elicited high levels of local and systemic immune responses in the animals during the first week, these effects were tapered off more quickly in the CLEC-treated group. Overall, the in vivo porcine data point to the potential of CLECs as a non-native and safe source of cells to treat cutaneous wounds.

Stat3 Tyrosine 705 and Serine 727 Phosphorylation Associate With Clinicopathological Characteristics and Distinct Tumor Cell Phenotypes in Triple-Negative Breast Cancer.

Signal transducer and activator of transcription 3 (Stat3) is responsible for many aspects of normal development and contributes to the development and progression of cancer through regulating epithelial cell identity and cancer stem cells. In breast cancer, Stat3 is associated with triple-negative breast cancers (TNBC) and its function has been related to the activation of p63, itself a marker of basal-like TNBC and a master regulator of stem cell activities. Stat3 activation is controlled by dual phosphorylation at tyrosine 705 (pTyr705) and serine 727 (pSer727), although it is unclear whether these have equivalent effects, and whether they are related or independent events. To address these issues, we investigated Stat3 phosphorylation at the two sites by immunohistochemistry in 173 patients with TNBC. Stat3 phosphorylation was assessed by automated quantitative measurements of digitized scanned images and classified into four categories based on histoscore. The results were analyzed for associations with multiple markers of tumor phenotype, proliferation, BRCA status, and clinicopathological characteristics. We show that the levels of pTyr705- and pSer727-Stat3 were independent in 34% of tumors. High pTyr705-Stat3 levels were associated with the luminal differentiation markers ERβ/AR and MUC1, whereas tumors with high levels of pSer727-Stat3 were more likely to be positive for the basal marker CK5/6, but were independent of p63 and were EGFR negative. Combined high pSer727- and low Tyr705-Stat3 phosphorylation associated with basal-like cancer. Although high Stat3 phosphorylation levels were associated with less aggressive tumor characteristics, they did not associate with improved survival, indicating that Stat3 phosphorylation is an unfavorable indicator for tumors with an otherwise good prognosis according to clinicopathological characteristics. These findings also show that pTyr705-Stat3 and pSer727-Stat3 associate with specific breast tumor phenotypes, implying that they exert distinct functional activities in breast cancer.

Sub-Classification of Triple-Negative Breast Cancer using Androgen Receptor and Cytokeratin 5/6

Background: Triple negative breast cancer (TNBC) is a unique heterogenous subtypes of breast cancer which is characterized by negative estrogen, progesterone, and human epidermal growth factor receptor (HER-2) status. TNBC displays different molecular phenotype with which basal-like tumour can be identified using high molecular weight basal cytokeratin 5/6 (CK5/6).
 Methods: Ninety-five (95) formalin fixed cases from Korle Bu Teaching Hospital in Ghana’s (KBTH) archives were sampled in a retrospective study from 2012-2016. Blocks of these triple-negative breast cancer was subclassified using CK5/6 and Androgen Receptor (AR) antibodies. Subclasses were also identified.
 Results and Conclusion: In all ninety-five (95) TNBC cases, hormonal subtyping was sub-classified using CK 5/6 and AR. The mean ±SD of these cases was recorded as 53.96 (±13.56) years and the age range of these cases was 22-104 years. The average size (±SD) of the tumour was recorded to be 14.43(±7.62) and it had a range of 2.4-45cm. lymph nodes retrieved also had a mean ± SD of 10.35(±6.05) with an average tumour lymph nodes involvement of 2.6(± 3.697).
 Invasive Ductal carcinoma was identified as the commonest histologic type of TNBC with approximately 95% of the cases. This was followed by invasive lobular (2.1%), medullary carcinoma (2.1%) and metaplastic carcinoma (1.1%).
 Approximately 30% of TNBC stained positive for CK5/6.
 It can however be concluded that, most TNBC are not basal-like when the basal marker CK5/6 is used.

Expansion of mouse castration-resistant intermediate prostate stem cells in vitro

BackgroundMost castration-resistant prostate cancers (CRPCs) have a luminal phenotype with high androgen receptor (AR) and prostate-specific antigen (PSA) expression. Currently, it is difficult to culture castration-resistant luminal cells with AR and PSA expression.MethodsWe formulated a custom-made medium and isolated primary cells from the prostate of adult wild-type (WT) and TRAMP mice. The cells were characterized by immunofluorescence staining, transcriptomic analysis, and qRT-PCR verification. Their self-renewal and differentiation potential in vitro and in vivo were examined. We treated the cells with androgen deprivation and enzalutamide and performed immunofluorescence staining and western blotting to analyze their expression of AR and PSA.ResultsWe isolated a novel type of castration-resistant intermediate prostate stem cells (CRIPSCs) from adult WT and TRAMP mice. The mouse CRIPSCs proliferated rapidly in two-dimensional (2D) culture dishes and can be cultured for more than six months. The mouse CRIPSCs expressed luminal markers (AR, PSA, and Dsg4), basal markers (CK5 and p63), Psca, and the intermediate cell marker (Ivl). Transcriptomic analysis showed that the mouse CRIPSCs had upregulated signaling pathways related to cancer development and drug resistance. In the long-term culture, TRAMP CRIPSCs had higher expression of the genes related to stem cells and cancers than WT mice. Both WT and TRAMP CRIPSCs formed organoids in Matrigel. WT CRIPSCs did not form prostate tissues when transplanted in vivo without urogenital sinus mesenchyme (UGM) cells. In contrast, TRAMP CRIPSCs formed prostate ducts in NOG mice without UGM cells and differentiated into luminal, basal, and neuroendocrine cells. Androgens regulated AR translocation between the nucleus and cytoplasm in the mouse CRIPSCs. Treatment of androgen deprivation (ADT) and enzalutamide reduced AR expression in WT and TRAMP CRIPSCs; however, this treatment promoted PSA expression in TRAMP, while not WT CRIPSCs, similar to the clinical observations of CRPC.ConclusionsOur study established a method for isolating and expanding mouse CRIPSCs in 2D culture dishes. Mouse CRIPSCs had markers of basal and luminal cells, including AR and PSA, and can differentiate into prostate organoids and tissues. TRAMP CRIPSCs had elevated PSA expression upon ADT and enzalutamide treatment. Our method can be translated into clinical settings for CRPC precision medicine.

Intratumoral Switch of Molecular Phenotype and Overall Survival in Muscle Invasive Bladder Cancer.

Simple SummaryThe Cancer Genome Atlas (TCGA) and more recent genome profiling recently revealed major intrinsic molecular subtypes in urothelial carcinoma (UC). Here we propose a fast and standardized immunophenotypical classification score (Piescore) that may discriminate between luminal, basal, or neu-like UC as a surrogate of molecular profile, and we describe, for the first time, an intratumoral phenotypical switch in tissue protein expression, from non-muscle to muscle-invasive progression. Our data show that a change from a luminal to a neu-like phenotype could worsen overall survival compared with a transition to a basal phenotype.In recent years, immunohistochemical protein expression was studied as a surrogate to the molecular classification of bladder cancer, although no tissue biomarkers are available for clinical use to predict survival or the response to neoadjuvant chemotherapy (CT) in UC, as the literature produced conflicting results. This retrospective study included TURB specimens harboring foci of HG pT2 muscle-invasive bladder carcinoma (MIBC) from 251 patients who subsequently underwent radical cystectomy. We performed immunohistochemical analysis on tumor samples, for relevant gene-expression-based markers for basal type (CD44, CK5/6) and luminal type (CK20 and pPARγ). Piescore, investigated in both non-muscle-invasive (NMI) and muscle-invasive (MI) components of the tumor, divided basal and luminal UC-types when at least three of the four markers were consistent with a specific phenotype, mixed types if one/two luminal and basal markers were present simultaneously, and neu-like types when all four markers investigated were negative. Eighteen selected cases were also investigated with RT-PCR to validate, and to increase the specificity of, the immunohistochemical results. We observe an immunophenotypical difference in the NMI and MI components in 96/251 UC patients (38.25%): half of tumors (44/96 cases) have a transition to basal, 36.46% (35/96 cases) to neu-like, 12.5% (12/96 cases) to mixed, and 5.2% (5/96 cases) to luminal phenotypes. Mixed tumors in the NMI component are more likely to change phenotype than other groups, particularly compared with basal tumors, which demonstrate greater stability (only 8/96 cases, p < 0.00001). The transition of luminal tumors to basal display a better OS compared with the transition toward neu-like tumors (p = 0.027). Overall, the phenotypical switch does not affect lymphovascular invasion, pT, DFS, or OS compared with non-switched cases. In the MI component, the presence of CD44 expression, irrespective of score-related phenotype, shows a protective effect in papillary-type UC (OS p = 0.008, HR 0.453, PFS p = 0.07, HR 0.599), and in UC naïve for CT (p = 0.0479). Piescore immunophenotyping reveals an intratumoral phenotypical transition between the NMI and MI components of the same tumor. The molecular change is a common event in the mixed and luminal categories, but not in basal tumors, which show better phenotypical stability. This phenomenon could partially explain the sensitivity of a subset of luminal UC to chemotherapy: good responders could be “non-real” luminal UC, which acquire nasal markers, such as CD44.