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18 pages, 7504 KiB  
Article
Pathophysiology, Histopathology, and Differential Diagnostics of Basal Cell Carcinoma and Cutaneous Squamous Cell Carcinoma—An Update from the Pathologist’s Point of View
by Iuliu Gabriel Cocuz, Maria Cătălina Popelea, Raluca Niculescu, Andrei Manea, Adrian-Horațiu Sabău, Andreea-Cătălina Tinca, Andreea Raluca Szoke, Corina Eugenia Budin, Adina Stoian, Silviu Horia Morariu, Titiana Cornelia Cotoi, Maria-Elena Cocuz and Ovidiu Simion Cotoi
Int. J. Mol. Sci. 2024, 25(4), 2220; https://doi.org/10.3390/ijms25042220 - 13 Feb 2024
Cited by 3 | Viewed by 4189
Abstract
Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) are the most frequently occurring non-melanocytic skin cancers. The objective of our study is to present the pathophysiology of BCC and cSCC and its direct relationship with the histopathological diagnostics and the differential [...] Read more.
Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) are the most frequently occurring non-melanocytic skin cancers. The objective of our study is to present the pathophysiology of BCC and cSCC and its direct relationship with the histopathological diagnostics and the differential diagnostics of these types of cancer, based on the morphological characteristics, immunohistochemical profile, and genetic alterations. The qualitative study was based on emphasizing the morphological characteristics and immunohistochemistry profiles of BCC and cSCC and the differential diagnostics based on the tissue samples from the Clinical Pathology Department of Mures Clinical County Hospital between 2020 and 2022. We analyzed the histopathological appearances and immunohistochemical profiles of BCC and cSCC in comparison with those of Bowen disease, keratoacanthoma, hyperkeratotic squamous papilloma, metatypical carcinoma, pilomatricoma, trichoblastoma, Merkel cell carcinoma, pleomorphic dermal sarcoma (PDS), and melanoma. Our study showed the importance of the correct histopathological diagnosis, which has a direct impact on the appropriate treatment and outcome for each patient. The study highlighted the histopathological and morphological characteristics of NMSCs and the precursor lesions in HE and the immunohistochemical profile for lesions that may make the differential diagnosis difficult to establish. Full article
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Figure 1
<p>Basal cell carcinoma—nodular (solid) type. (<b>A</b>). Tegument covered by a keratinized stratified squamous epithelium, showing a tumoral proliferation in the underlying dermis composed of plaques of cells with a basaloid appearance, with nuclei palisading at the periphery. The plaques have a solid, compact appearance, with varied shapes and sizes. (<b>B</b>). Keratinized stratified squamous epithelium, ulcerated, showing a tumoral proliferation in the underlying dermis composed of plaques of cells with a basaloid appearance with abundant chronic inflammatory infiltrate in the peritumoral stroma.</p>
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<p>Basal cell carcinoma—superficial multicentric type. (<b>A</b>). Tegument covered by a keratinized, stratified squamous epithelium, ulcerated, intermittently presenting nests of basaloid-type tumor cells at the base of the epidermis with basophilic, reduced cytoplasm, large, hyperchromatic nuclei, and a palisade arrangement of nuclei at the periphery. (<b>B</b>). Ulcerated epidermis intermittently presenting nests of basaloid-type tumor cells at the base with peritumoral artifact retraction and an abundant chronic inflammatory infiltrate.</p>
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<p>Basal cell carcinoma—fibroepithelial (Pinkus) type. (<b>A</b>). Interconnected strands and clusters of basaloid cells enveloped by a fibrous stroma. When viewed from a three-dimensional standpoint, the tumor takes on the appearance of a honeycomb or sponge, characterized by thin epithelial septa, with the intervening spaces filled by stroma. Along the edges of the fenestrations, columnar cells are organized in a palisade formation. The tumor cells are positive for anti-CK AE1/AE3 (<b>B</b>) and anti-Bcl-2 antibodies (<b>C</b>).</p>
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<p>Basal cell carcinoma—adenoid and cystic type, infiltrative type, and micronodular type. (<b>A</b>). Adenoid and cystic type. Tumoral proliferation composed of plaques with pseudo-glandular appearance or with cystic degeneration. At the periphery of the plaques, nuclear palisading and artifact retraction of the surrounding stroma are observed. (<b>B</b>). Infiltrative type. Cords and trabeculae of basaloid-type tumor cells with basophilic, reduced cytoplasm, large nuclei, hyperchromatism, and a disposition resembling nuclear palisading at the periphery. The intertumoral stroma exhibits an abundant lymphoplasmacytic inflammatory infiltrate. (<b>C</b>). Micronodular type. Skin fragment covered by keratinized stratified squamous epithelium, presenting a tumoral proliferation in the underlying dermis composed of small, variable-shaped plaques with a solid, basaloid appearance.</p>
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<p>Cutaneous squamous cell carcinoma—well-differentiated. (<b>A</b>,<b>B</b>). Skin fragment covered by keratinized, stratified, squamous epithelium, presenting a tumoral proliferation in the underlying dermis composed of plaques of tumor cells with a squamous appearance. The plaques have a solid, compact appearance, with varied shapes and sizes, exhibiting marked keratinization phenomena. Adjacent to the tumoral plaques, there is an abundant polymorphic inflammatory infiltrate. At a higher magnification, the tumor cells have abundant, eosinophilic cytoplasm; enlarged, pleomorphic nuclei; mitotic figures, along with keratin pearl (<b>C</b>) formations.</p>
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<p>Cutaneous squamous cell carcinoma—moderately differentiated. (<b>A</b>). Proliferation of irregular patches and plaques with polygonal squamous cells, exhibiting marked cyto-nuclear atypia with rare occurrences of keratinization. Around the tumor, an abundant, mixed, inflammatory infiltrate is observed. The tumor cells are positive for anti-CK AE1/AE3 antibodies (<b>B</b>) and for anti-p53 antibodies (<b>C</b>).</p>
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<p>Cutaneous squamous cell carcinoma—poorly differentiated. (<b>A</b>). Solid proliferation of polygonal squamous cells with marked cyto-nuclear atypia. The cells have an abundant cytoplasm, which is either eosinophilic or pale, large pleomorphic nuclei with eosinophilic nucleoli, and numerous atypical mitoses. No keratinization phenomena are observed. (<b>B</b>). The tumor cells are positive for anti-CK AE1/AE3 antibodies.</p>
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<p>Keratoacanthoma, hyperkeratotic squamous papilloma, and in situ squamous cell carcinoma (Bowen disease). (<b>A</b>). Keratoacanthoma. Tegument covered by a keratinized stratified squamous epithelium, composed of squamous cells with minimal atypia, exhibiting pronounced orthokeratotic hyperkeratosis phenomena. (<b>B</b>). Hyperkeratotic squamous papilloma. Keratinized stratified squamous epithelium, without atypia, presenting a papillomatous formation on the surface covered by a keratinized stratified squamous epithelium with marked orthokeratotic hyperkeratosis, along with an acanthotic spinous layer. (<b>C</b>). In situ squamous cell carcinoma (Bowen disease). Skin fragment covered by intact keratinized stratified squamous epithelium, exhibiting marked dysplasia throughout its thickness. Tumor cells display pleomorphism, enlarged nuclei, and irregular nuclear membranes, with numerous mitoses. The tumor cells do not infiltrate the underlying dermis.</p>
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<p>Metatypical carcinoma (basosquamous). (<b>A</b>). Tegument lined by a keratinized stratified squamous epithelium, exhibiting a tumoral proliferation consisting of two components: a predominantly squamous cell carcinoma component and a basal cell carcinoma component. (<b>B</b>). The tumor cells are positive for anti-CK AE1/AE3 antibodies in the BCC component. (<b>C</b>). The tumor cells are positive for anti-CK AE1/AE3 antibodies in the cSCC component.</p>
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<p>Pilomatricoma and trichoblastoma. (<b>A</b>). Pilomatricoma—tumoral proliferation consisting of islands of basaloid cells without atypia, occasionally flattened, and focal areas completely devoid of cells, replaced by cells with indistinct contours, intensely eosinophilic cytoplasm, and inconspicuous nuclei (phantom cells) extending towards the center of the lesion. (<b>B</b>). Trichoblastoma. Tumoral proliferation composed of nodules of varying sizes with cells that have basophilic cytoplasm and are monomorphic and without cyto-nuclear atypia. The surrounding stroma has a fibromyxoid appearance.</p>
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<p>Merkel cell carcinoma. (<b>A</b>,<b>B</b>). The tumor formation exhibits a solid architecture characterized by plaques and trabeculae of monomorphic cells, small to medium in size. The tumor cells do not show epidermotropism, without an ulceration of the epidermis. Between the tumoral plaques, there is a moderate chronic inflammatory infiltrate. (<b>C</b>). At a higher magnification, the tumoral cells have a hyperchromatic, centrally located nucleus with granular and dispersed chromatin, along with an elevated mitotic index. Tumor cells are positive for anti-CK-20 antibodies (<b>D</b>,<b>E</b>), anti-CD56 antibodies (<b>F</b>), anti-Chromogranin A antibodies (<b>G</b>), anti-Synaptophysin antibodies (<b>H</b>), and anti-NSE antibodies (<b>I</b>).</p>
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<p>Pleomorphic dermal sarcoma (PDS). (<b>A</b>). Tumoral proliferation with solid architecture composed of pleomorphic tumoral cells with epithelioid or fusiform appearances, vesicular nuclei, and prominent nucleoli admixed with multinucleated giant cells, along with numerous mitotic figures. Tumor cells are positive for anti-Vimentin antibodies (<b>B</b>) and anti-CD68 antibodies (<b>C</b>).</p>
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<p>Nodular melanoma. (<b>A</b>). Tumoral cells with epithelioid appearances with abundant eosinophilic cytoplasm and vesicular nuclei and prominent nucleoli. Tumor cells are positive for anti-SOX10 antibodies (<b>B</b>) and anti-HMB45 (red chromogen) antibodies (<b>C</b>).</p>
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11 pages, 46592 KiB  
Article
TRPS1: A Marker of Follicular Differentiation
by Kristin J. Rybski, Hatice B. Zengin and Bruce R. Smoller
Dermatopathology 2023, 10(2), 173-183; https://doi.org/10.3390/dermatopathology10020025 - 14 Jun 2023
Cited by 3 | Viewed by 2889
Abstract
The trichorhinophalangeal syndrome type 1 (TRPS1) immunohistochemical (IHC) stain has increased in use in recent years as a marker for breast carcinomas. The TRPS1 gene is involved in various tissues, including the growth and differentiation of hair follicles. This article seeks to evaluate [...] Read more.
The trichorhinophalangeal syndrome type 1 (TRPS1) immunohistochemical (IHC) stain has increased in use in recent years as a marker for breast carcinomas. The TRPS1 gene is involved in various tissues, including the growth and differentiation of hair follicles. This article seeks to evaluate the IHC expression of TRPS1 in cutaneous neoplasms with follicular differentiation, such as trichoblastoma (TB), trichoepithelioma (TE), and basal cell carcinoma (BCC). IHC studies were performed on 13 TBs, 15 TEs, and 15 BCCs with an antibody against TRPS1. The study found a variable staining expression of TRPS1 in the tumor nests of TB, TE, and BCC. BCCs were distinct in that none of the BCCs demonstrated intermediate or high positivity, while TBs and TEs showed intermediate-to-high positivity in 5/13 (38%) and 3/15 (20%) of cases, respectively. We observed a distinct staining pattern among the mesenchymal cells of TB and TE. We found that TRPS1 highlighted perifollicular mesenchymal cells adjacent to the nests of TB and TE tumor cells. This staining pattern was absent in BCCs, where only scattered stromal cells were positive for TRPS1. Papillary mesenchymal bodies were also highlighted by TRPS1 in TB and TE. TRPS1 stained various parts of the normal hair follicle, including the nuclei of cells in the germinal matrix, outer root sheaths, and hair papillae. TRPS1 may be a useful IHC marker for follicular differentiation. Full article
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<p>Examples of TRPS1 immunoreactivity scores in the tumor nests. (<b>a</b>) Negative TRPS1 score in trichoepithelioma and positive staining of mesenchymal cells (200×); (<b>b</b>) low positive TRPS1 score in basal cell carcinoma (200×); (<b>c</b>) intermediate positive TRPS1 score in trichoepithelioma (200×); (<b>d</b>) high positive TRPS1 score in trichoblastoma (200×).</p>
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<p>Examples of TRPS1 immunoreactivity scores in the tumor nests. (<b>a</b>) Negative TRPS1 score in trichoepithelioma and positive staining of mesenchymal cells (200×); (<b>b</b>) low positive TRPS1 score in basal cell carcinoma (200×); (<b>c</b>) intermediate positive TRPS1 score in trichoepithelioma (200×); (<b>d</b>) high positive TRPS1 score in trichoblastoma (200×).</p>
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<p>TRPS1 IHC highlighting perifollicular mesenchymal cells in trichoblastoma. (<b>a</b>) Trichoblastoma (H&amp;E, 100×); (<b>b</b>,<b>c</b>) TRPS1 strongly stained mesenchymal cells surrounding the tumor nests in trichoblastoma (100× and 200×).</p>
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<p>TRPS1 IHC demonstrating scattered mesenchymal cells in basal cell carcinoma. (<b>a</b>) Basal cell carcinoma (H&amp;E, 100×); (<b>b</b>,<b>c</b>) TRPS1 stains scattered mesenchymal cells in basal cell carcinoma (100× and 200×).</p>
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<p>TRPS1 IHC demonstrating scattered mesenchymal cells in basal cell carcinoma. (<b>a</b>) Basal cell carcinoma (H&amp;E, 100×); (<b>b</b>,<b>c</b>) TRPS1 stains scattered mesenchymal cells in basal cell carcinoma (100× and 200×).</p>
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<p>TRPS1 IHC staining papillary mesenchymal bodies in trichoepithelioma. (<b>a</b>,<b>b</b>) Trichoepithelioma with papillary mesenchymal bodies highlighted by the TRPS1 stain (H&amp;E and TRPS1, 200×).</p>
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<p>TRPS1 IHC expression in normal hair follicles. (<b>a</b>) Normal hair follicle (H&amp;E, 100×); (<b>b</b>) TRPS1 staining the root sheaths, germinative cells, matrix cells, and dermal papilla of a hair follicle (100×).</p>
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<p>(<b>a</b>) Trichoblastoma (H&amp;E, 100×). (<b>b</b>) High positive TRPS1 immunoreactivity in the trichoblastoma tumor nests (100×).</p>
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<p>(<b>a</b>) Trichoepithelioma with a papillary mesenchymal body in the lower right corner (H&amp;E, 100×). (<b>b</b>) High positive TRPS1 immunoreactivity in trichoepithelioma tumor nests and the papillary mesenchymal body (100×).</p>
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<p>(<b>a</b>) Basal cell carcinoma with characteristic clefting (H&amp;E, 100×). (<b>b</b>) Negative TRPS1 immunoreactivity in BCC tumor nests (100×).</p>
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13 pages, 1850 KiB  
Article
Significance of Dermoscopy in Association with Clinical Features in Differentiation of Basal Cell Carcinoma and Benign Trichoblastic Tumours
by Martyna Sławińska, Anna Płaszczyńska, Joanna Lakomy, Krzysztof Pastuszak, Wojciech Biernat, Monika Sikorska, Roman J. Nowicki and Michał Sobjanek
Cancers 2022, 14(16), 3964; https://doi.org/10.3390/cancers14163964 - 17 Aug 2022
Cited by 4 | Viewed by 5282
Abstract
Background: Although basal cell carcinoma (BCC) can, in the majority of cases, be diagnosed based on clinical and dermoscopic assessment, a potential overlap with benign adnexal skin tumours seems to exist, including trichoblastic tumours (TT). Methods: Retrospective analysis of clinical and dermoscopic [...] Read more.
Background: Although basal cell carcinoma (BCC) can, in the majority of cases, be diagnosed based on clinical and dermoscopic assessment, a potential overlap with benign adnexal skin tumours seems to exist, including trichoblastic tumours (TT). Methods: Retrospective analysis of clinical and dermoscopic features of benign TT and BCC cases was performed to develop a diagnostic algorithm with a potential utility in clinical practice. Results: In the study, 502 histopathologically confirmed BCC cases were compared with 61 TT (including 44 TB (72.13%), 10 TE (16.39%) and 7 DTE (11.48%]). Patients in the BCC group were statistically older (mean age was 71.4 vs. 64.4 years, respectively; p = 0.009). BCC presented generally as larger tumours (mean tumour size 11.0 vs. 8.2 mm for the TT group; p = 0.001) and was more frequently associated with clinically visible ulceration (59.4% vs. 19.7%, respectively; p < 0.001). Comparison of lesion morphology, clinically visible pigmentation, and anatomical location did not show significant differences between the analysed groups. Dermoscopically visible ulceration was significantly more common in the BCC group compared to the TT group (52.2% vs. 14.8%; p < 0.0001). Pigmented structures, specifically brown dots and brown globules, were significantly more prevalent in the TT group (32.8% vs. 11.4%; p = 0.0001 and 29.5% vs. 8.2%; p <0.0001). Similarly, TT more commonly than BCC showed the presence of cloudy/starry milia-like cysts (26.2% vs. 11.6%; p = 0.0031) and yellow globules (16.4% vs. 7.2%; p = 0.033). Conclusions: Despite differences in frequency of clinical and dermoscopic features between BCC and TT in the studied group, differential diagnosis based on these variables is not reliable. Histopathological examination remains a diagnostic gold standard in differentiation of BCC and TT. Full article
(This article belongs to the Section Cancer Causes, Screening and Diagnosis)
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<p>Two clinically similar tumours presenting as whitish plaque within the cheek area. (<b>a</b>,<b>b</b>) Desmoplastic trichoepithelioma; dermoscopy shows branched (arborizing) vessels (red arrow) over whitish background (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification). (<b>c</b>,<b>d</b>) Morpheaform basal cell carcinoma; dermoscopy shows central ulceration (black arrow) and branched (arborizing) vessels (red arrow) over whitish background, starry milia-like cysts (white arrow) and Multiple Aggregated Yellow-White Globules (yellow arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification).</p>
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<p>Two clinically similar tumours presenting as non-ulcerated, partially pigmented nodules within the lower eyelid region. (<b>a</b>,<b>b</b>) Trichoblastoma; dermoscopy shows branched (arborizing) vessels over whitish background (red arrow), superficial fine telangiectasia (violet arrow), brown dots and globules (brown arrow), yellow globules (yellow arrow), and starry milia-like cysts (white arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification). (<b>c</b>,<b>d</b>) Nodular basal cell carcinoma; dermoscopy shows branched (arborizing) vessels (red arrow), blue-grey globules (grey arrow), brown dots and globules (brown arrow), starry milia-like cysts (white arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification).</p>
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<p>Two clinically similar, partially pigmented tumours within the ala nasi. (<b>a</b>,<b>b</b>) Trichoblastoma; dermoscopy shows branched (arborizing) vessels (red arrow), ulcerations (black arrow), large blue ovoid nests (blue arrow), yellow globules (yellow arrow), and starry milia-like cysts (white arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification). (<b>c</b>,<b>d</b>) Nodulo-infiltrative basal cell carcinoma; dermoscopy shows branched (arborizing) vessels (red arrow), blue-grey globules (grey arrow), and large blue ovoid nest (blue arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification).</p>
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<p>Two clinically similar tumours in the area of left medial eye canthus. (<b>a</b>,<b>b</b>) Trichoepithelioma; dermoscopy shows branched (arborizing) vessels (red arrow), superficial fine telangiectasia (violet arrow), central ulceration (black arrow), brown dots and globules (brown arrow), yellow globules (yellow arrow), and starry milia-like cysts (white arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification). (<b>c</b>,<b>d</b>) Nodular basal cell carcinoma; dermoscopy shows branched (arborizing) vessels (red arrow), brown dots (brown arrow), starry milia-like cysts (white arrow), yellow globules (yellow arrow), and peripheral brown structureless areas (green arrow) (Fotofinder Vexia; Camera Medicam 800 HD, ×20 magnification).</p>
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<p>(<b>a</b>) ROC curve of the classification algorithm tested against the independent test set; (<b>b</b>) prediction scores for each sample from the independent test set. The scores may be interpreted as the similarity of the particular sample with the samples from the training set. The final classification would depend on the assumed cut-off level. The closer the score is to 0, the more the sample resembles basal cell carcinoma (BCC). On the left, samples for which the real diagnosis is BCC. On the right, samples with trichoblastic tumours (TT). (<b>c</b>) Feature importance of the final classifier, as measured on the training set using built-in XGBoost method. (<b>d</b>) The final algorithm tree. The result is a number in (0.1) range which is later transformed into binary decision based on the determined cut-off. Please note that the features were coded as numeric variables, hence “&gt;0.5” in case of binary features actually means the presence of the feature.</p>
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16 pages, 10578 KiB  
Article
Cutaneous and Subcutaneous Tumours of Small Pet Mammals—Retrospective Study of 256 Cases (2014–2021)
by Iwona Otrocka-Domagała, Katarzyna Paździor-Czapula, Joanna Fiedorowicz, Mateusz Mikiewicz, Agnieszka Piotrowska and Michał Gesek
Animals 2022, 12(8), 965; https://doi.org/10.3390/ani12080965 - 8 Apr 2022
Cited by 15 | Viewed by 4557
Abstract
Since small mammals are gaining popularity as pets in Poland, the number of tumour samples submitted for histopathological examination is quite high. This study was a retrospective analysis of cutaneous and subcutaneous tumours in small pet mammals submitted for histopathology in 2014–2021. The [...] Read more.
Since small mammals are gaining popularity as pets in Poland, the number of tumour samples submitted for histopathological examination is quite high. This study was a retrospective analysis of cutaneous and subcutaneous tumours in small pet mammals submitted for histopathology in 2014–2021. The analysis included 256 tumours sampled from 103 guinea pigs, 53 rats, 43 pet rabbits, 21 ferrets, 17 hamsters, 8 degus, 5 African pygmy hedgehogs, 3 Mongolian gerbils and 3 chinchillas. Tumours were diagnosed based on routine histopathology, with additional immunohistochemistry when necessary. The results of this study revealed that the vast majority of cutaneous tumours in guinea pigs were benign, with a predominance of lipoma. Adnexal tumours constituted a significant percentage of cutaneous tumours in guinea pigs (24.3%, with the most common being trichofolliculoma), pet rabbits (46.5%, with the most common being trichoblastoma), ferrets (33.3%, mostly derived from sebaceous glands), hamsters (52.9%, with the most common being trichoepithelioma) and gerbils (66.7%, scent gland epithelioma). Soft tissue sarcomas were a predominant group of tumours in rats (52.8%, with the most common being fibrosarcoma), African pygmy hedgehogs (100%), degus (87.5%) and chinchillas (66.7%). Melanocytic tumours were only sporadically seen in small mammal pets. Mast cell tumours were diagnosed only in ferrets, while epitheliotropic T-cell lymphoma was diagnosed only in a hamster and a degu. In summary, malignant tumours constitute a significant percentage of cutaneous tumours in many species of small mammal pets. Therefore, each cutaneous tumour should be sampled for further cytologic or histopathologic diagnosis. Full article
(This article belongs to the Special Issue Advances in Veterinary Oncology)
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<p>Histiocytic sarcoma, submandibular area, rat. Pleomorphic tumour cells are arranged haphazardly and show mitotic figures (arrows). HE. Inset: tumour cells express Iba-1. IHC.</p>
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<p>Compound melanocytoma, facial area, pet rabbit. Heavily melanised, well-differentiated tumour cells formed solid sheets and massively infiltrated the epidermis. HE.</p>
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<p>Poorly differentiated sarcoma, tail, ferret. Tumour cells show high anisocytosis and anisokaryosis and undergo necrosis (arrows). HE. Upper inset: tumour cells show cytoplasmic expression of vimentin. IHC. Lower inset: tumour cells show slight cytoplasmic expression of α-SMA. IHC.</p>
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<p>T-cell epitheliotropic lymphoma, tail, golden hamster. Large lymphocytes massively infiltrated the skin and adnexa, and there is a single entrapped sebaceous gland visible (arrow). HE. Inset: tumour cells show expression of CD3. IHC.</p>
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<p>Anaplastic sarcoma with giant cells, neck, degu. The tumour cells show high levels of anaplasia and multinucleation. HE. Inset: tumour cells show cytoplasmic expression of vimentin. IHC.</p>
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<p>Histiocytic sarcoma, forelimb, African pygmy hedgehog. Pleomorphic tumour cells show high anisocytosis and anisokaryosis. Nuclei are large with marginated chromatin and distinct, single nucleoli. HE. Inset: tumour cells show expression of Iba-1. IHC.</p>
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<p>Fibrosarcoma, area unspecified, chinchilla. There are bundles of spindle to oval tumour cells and moderate amount of collagen fibres between them. Tumour cells undergo focal necrosis (arrow). HE.</p>
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<p>Epithelioid haemangioma, facial (nasal) area, chinchilla. Large and plump tumour cells form small blood vessels and solid aggregates. HE.</p>
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12 pages, 4598 KiB  
Article
The High Expression of Legumain in Canine Neoplasms: A Retrospective Analysis of 100 Cases
by Chiao-Hsu Ke, Ka-Mei Sio, Shang-Lin Wang, Ying Kuo, Wei-Hsiang Huang and Chen-Si Lin
Animals 2022, 12(4), 504; https://doi.org/10.3390/ani12040504 - 17 Feb 2022
Cited by 5 | Viewed by 2479
Abstract
Legumain, a novel asparaginyl endopeptidase, has been observed to be overexpressed in several types of human solid tumors. Elevated levels of legumain are found in human cancers, and this oncoprotein may facilitate tumor invasion and metastasis when overexpressed. These findings suggest that legumain [...] Read more.
Legumain, a novel asparaginyl endopeptidase, has been observed to be overexpressed in several types of human solid tumors. Elevated levels of legumain are found in human cancers, and this oncoprotein may facilitate tumor invasion and metastasis when overexpressed. These findings suggest that legumain plays a malignant role in cancer biology. However, currently, no publications have identified the role of legumain in the development of canine cancers. The present study first compared the expression patterns of legumain in paraffin-embedded canine tumor tissues, with those of normal tissues, by immunohistochemistry. A total of 100 canine tumor samples, including mast cell tumors, soft tissue sarcoma, hemangiosarcoma, lymphoma, mammary gland carcinoma, hepatoid gland tumor, squamous cell carcinoma, trichoblastoma, and melanoma were evaluated. Compared with the normal tissues, all tumor samples displayed high intensities of legumain expression. Mesenchymal-type tumors displayed immunoreactivity for legumain, with an average expression of 40.07% ± 1.70%, which was significantly lower than those of epithelial tumors and other types of tumors, which had median expressions of 49.12% ± 1.75% and 47.35% ± 2.71%, respectively (p < 0.05). These findings indicate that legumain has a high potential to be a candidate for distinguishing tumors from normal tissues. Although further studies on a larger number of cases are necessary to clarify the clinical application of legumain, the overexpression patterns of legumain in canine tumor tissues are reported, for the first time, in this study. Full article
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<p>Western blot analysis of the specificity of anti-legumain polyclonal antibody in different canine cancer cell lines. Representative canine cell lines, including canine melanoma (M5 and KMec), mammary gland tumor (CMT-1 and MPG), and lymphoma (UL-1 and CLBL-1), were selected. Protein lysate of M5, KMec, CMT-1, MPG, UL-1, and CLBL-1 cell lines are shown. All the samples presented a dominant band near 54 kDa, indicating that this antibody can identify the corresponding proteins in canine species.</p>
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<p>Expression patterns of legumain in canine normal tissues. Relatively low expression levels of legumain in these tissues, including (<b>a</b>) representative samples of the skin, thymus, muscle, and nerve. (<b>b</b>) Statistical analysis of legumain expression among different tissues. All data are expressed as mean ± SEM (<span class="html-italic">n</span> = 5).</p>
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<p>Expression patterns of legumain in canine normal tissues. Relatively low expression levels of legumain in these tissues, including (<b>a</b>) representative samples of the skin, thymus, muscle, and nerve. (<b>b</b>) Statistical analysis of legumain expression among different tissues. All data are expressed as mean ± SEM (<span class="html-italic">n</span> = 5).</p>
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<p>Statistical analysis of legumain expression in different types of canine tumors. Significantly elevated patterns were found in these tumors. Data are shown as mean ± SEM (<span class="html-italic">n</span> = 5–10). MCT, mast cell tumor; HAS, hemangiosarcoma; STS, soft tissue sarcoma; SCC, squamous cell carcinoma; MGT, mammary gland tumor (carcinoma); HGT, hepatoid gland tumor; SM, skin melanoma; OM, oral melanoma.</p>
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<p>Immunochemistry staining of legumain in mesenchymal tumors. Overexpression of legumain was found in (<b>a</b>) low-grade MCT; (<b>b</b>) high-grade MCT; (<b>c</b>) lymphoma. Increased expression of legumain was found in the (<b>d</b>) HAS and (<b>e</b>) STS. Representative samples are shown and five high power fields (40× objective magnification) per tumor (<span class="html-italic">n</span> = 5–10) were randomly selected and scored. The bar represents 20 µm. MCT, mast cell tumor; HAS, hemangiosarcoma; STS, soft tissue sarcoma.</p>
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<p>Legumain signal determined by immunochemistry. Elevated levels of legumain were found in non-mesenchymal tumors, including (<b>a</b>) squamous cell carcinoma; (<b>b</b>) mammary gland carcinoma; (<b>c</b>) hepatoid gland tumor; and (<b>d</b>) trichoblastoma. (<b>e</b>) Oral melanoma and (<b>f</b>) skin melanoma displayed heightened legumain expression. Representative samples are shown and five high power fields (40× objective magnification) per tumor sample (<span class="html-italic">n</span> = 5–10) were randomly selected and scored. The bar represents 20 µm.</p>
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<p>Statistical analysis of legumain expression among three tumor types. Legumain expressions in melanoma and epithelial tumors were higher than those in mesenchymal tumors. All data are expressed as mean ± SEM. Statistical significance was calculated with the Kruskal–Wallis test. * <span class="html-italic">p</span> &lt; 0.05.</p>
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6 pages, 1386 KiB  
Review
The Multiple Faces of Nodular Trichoblastoma: Review of the Literature with Case Presentation
by Gerardo Cazzato, Antonietta Cimmino, Anna Colagrande, Francesca Arezzo, Lucia Lospalluti, Sara Sablone, Teresa Lettini, Leonardo Resta and Giuseppe Ingravallo
Dermatopathology 2021, 8(3), 265-270; https://doi.org/10.3390/dermatopathology8030032 - 5 Jul 2021
Cited by 8 | Viewed by 6421
Abstract
Trichoblastoma (TB) is a rare biphasic benign adnexal neoplasm originating from follicular germ cells but clinically, it can simulate basal cell carcinoma (BCC), making the diagnosis more difficult. There are several variants of Trichoblastoma and a good knowledge of these is essential for [...] Read more.
Trichoblastoma (TB) is a rare biphasic benign adnexal neoplasm originating from follicular germ cells but clinically, it can simulate basal cell carcinoma (BCC), making the diagnosis more difficult. There are several variants of Trichoblastoma and a good knowledge of these is essential for correct diagnosis and management. We report two new cases observed in the last year at our Pathological Anatomy Operative Unit, and conduct a careful review of the literature, from the first description of this lesion by Headington in 1970 to the most recent classifications. Full article
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Figure 1
<p>Clinical features of the “Case 1” lesion.</p>
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<p>(<b>A</b>) Clinical features of the “Case 2” lesion, well circumscribed, symmetrical, smooth bordered, skin-colored pinkish or brown, at the level of the passage from the tip to the right nasal wing. (<b>B</b>) Dermoscopic features of TB with arborizing vessels and teangiectasias.</p>
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<p>Proliferation of numerous small and irregular nests of basaloid cells with no obvious connection to the epidermis (hematoxylin and eosin, original magnification, (<b>A</b>) 40× (<b>B</b>) 100×).</p>
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<p>Proliferation of large nests of basaloid cells without connection to the epidermis (hematoxylin and eosin, original magnification, (<b>A</b>) 40× (<b>B</b>) 100×).</p>
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8 pages, 2231 KiB  
Case Report
Histological and Immunohistochemical Features of Trichoblastoma in a Sarda Breed Sheep
by Marta Polinas, Giovanni P. Burrai, Veronica Vitiello, Laura Falchi, Maria T. Zedda, Gerolamo Masala, Vincenzo Marras, Giulia Satta, Alberto Alberti and Elisabetta Antuofermo
Animals 2020, 10(11), 2039; https://doi.org/10.3390/ani10112039 - 4 Nov 2020
Cited by 3 | Viewed by 3712
Abstract
Skin tumors with adnexal differentiation are commonly reported in dogs and cats, while only anecdotal evidence is available in sheep. Here we illustrate the macroscopic, histologic, and immunohistochemical features of a cutaneous lesion with adnexal differentiation in a 6-year-old female Sarda breed sheep, [...] Read more.
Skin tumors with adnexal differentiation are commonly reported in dogs and cats, while only anecdotal evidence is available in sheep. Here we illustrate the macroscopic, histologic, and immunohistochemical features of a cutaneous lesion with adnexal differentiation in a 6-year-old female Sarda breed sheep, surgically treated for a horn-like mass located in the left pinna. Additionally, we investigate a possible contribution of Ovine Papillomaviruses (OaPVs). Histologically, the dermis was expanded by an expansive and unencapsulated multilobulated nodule composed of cuboidal to spindle basaloid cells arranged in variably-sized cytokeratins (CK) AE1-AE3, CK 5/6 and CK 34 beta E12, p63—positive winding cords with a characteristic palisade arrangement of neoplastic cells in the periphery of the tumor. Based on these results, the cutaneous neoplasm was diagnosed as a trabecular trichoblastoma with spindle cells and rare structures resembling papillary mesenchymal bodies. Additionally, multiple enlarged sebaceous lobules clustered around dilated ducts suggestive of sebaceous gland hyperplasia were detected near the trichoblastoma. No PV DNA was found in the examined tissues, suggesting that ovine PVs are not involved in the pathogenesis of the present skin tumors with adnexal differentiation. Further investigations and efforts are required to elucidate the prevalence of skin tumors with adnexal differentiation in this species. Full article
(This article belongs to the Special Issue Spontaneous Neoplasms in Animals)
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<p>Left pinna: horn-like exophytic cutaneous mass.</p>
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<p>Haired skin: cut section of the cutaneous mass located in the left pinna. Two distinct multilobulated masses circumscribed by multiple white-yellowish bands and measuring 3 × 1.2 × 1 cm (arrow) and 1.3 × 1.2 × 0.5 cm (asterisk) are observed. Bar: 0.5 cm.</p>
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<p>(<b>A</b>) Columns and trabeculae of basaloid epithelial cells of the trichoblastoma. Hematoxylin and eosin (H&amp;E). Bar 10 µm. (<b>B</b>) Foci of mesenchymal cells closely associated with neoplastic epithelial cells resembling papillary mesenchymal bodies. H&amp;E. Bar 10 µm.</p>
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<p>Immunohistochemistry. DAB and hematoxylin (<b>A</b>) Cytokeratin 5/6 strong and diffuse cytoplasm immunostaining of epithelial basaloid trichoblastic cells. DAB and hematoxylin. Bar 10 µm; (<b>B</b>) P63 strong and diffuse nuclear expression of epithelial basaloid trichoblastic cells. Bar 10 µm; (<b>C</b>) Moderate and focal cytoplasmic immunoreactivity of CD56 was multifocally detected in 8–10% of epithelial basaloid trichoblastic cells. Bar 10 µm.</p>
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<p>Enlarged sebaceous lobules oriented around a small dilated duct lined by squamous keratinizing epithelium characterized the sebaceous gland hyperplasia. H&amp;E. Bar 50 µm.</p>
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20 pages, 4254 KiB  
Article
Merkel Cell Polyomavirus T Antigens Induce Merkel Cell-Like Differentiation in GLI1-Expressing Epithelial Cells
by Thibault Kervarrec, Mahtab Samimi, Sonja Hesbacher, Patricia Berthon, Marion Wobser, Aurélie Sallot, Bhavishya Sarma, Sophie Schweinitzer, Théo Gandon, Christophe Destrieux, Côme Pasqualin, Serge Guyétant, Antoine Touzé, Roland Houben and David Schrama
Cancers 2020, 12(7), 1989; https://doi.org/10.3390/cancers12071989 - 21 Jul 2020
Cited by 25 | Viewed by 3584
Abstract
Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). It is still under discussion, in which cells viral integration and MCC development occurs. Recently, we demonstrated that a virus-positive MCC derived from a trichoblastoma, an [...] Read more.
Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). It is still under discussion, in which cells viral integration and MCC development occurs. Recently, we demonstrated that a virus-positive MCC derived from a trichoblastoma, an epithelial neoplasia bearing Merkel cell (MC) differentiation potential. Accordingly, we hypothesized that MC progenitors may represent an origin of MCPyV-positive MCC. To sustain this hypothesis, phenotypic comparison of trichoblastomas and physiologic human MC progenitors was conducted revealing GLI family zinc finger 1 (GLI1), Keratin 17 (KRT 17), and SRY-box transcription factor 9 (SOX9) expressions in both subsets. Furthermore, GLI1 expression in keratinocytes induced transcription of the MC marker SOX2 supporting a role of GLI1 in human MC differentiation. To assess a possible contribution of the MCPyV T antigens (TA) to the development of an MC-like phenotype, human keratinocytes were transduced with TA. While this led only to induction of KRT8, an early MC marker, combined GLI1 and TA expression gave rise to a more advanced MC phenotype with SOX2, KRT8, and KRT20 expression. Finally, we demonstrated MCPyV-large T antigens’ capacity to inhibit the degradation of the MC master regulator Atonal bHLH transcription factor 1 (ATOH1). In conclusion, our report suggests that MCPyV TA contribute to the acquisition of an MC-like phenotype in epithelial cells. Full article
(This article belongs to the Special Issue The Biological and Clinical Aspects of Merkel Cell Carcinoma)
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Figure 1
<p>Merkel cells and possible Merkel cell progenitors in human skin. (<b>A</b>) Keratin 20 (KRT20), SRY-box transcription factor 2 (SOX2), KRT8, and KRT18 staining was used to identify Merkel cells (MCs) (bar = 100 µm) (only one hotspot investigated for illustration purpose). Merged analysis is available in <a href="#app1-cancers-12-01989" class="html-app">Figure S1B</a>. (<b>B</b>) Identification of potential MC progenitors in human skin: Three MC hotspots as well as interfollicular epidermis for comparison are depicted (bar = 100 µm) (15 hotspots investigated in total). Immunohistochemical staining revealed expression of KRT17 and SOX9 in the epidermal cells surrounding differentiated MCs suggesting that these cells are MC progenitors. Nuclear GLI family zinc finger 1 (GLI1) was detected only close to MC hotspots in hairy, but not in acral skin. Of note, neurofilament (NF)-expressing dermal nerves were observed in contact with the MCs.</p>
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<p>Ectopic GLI1 expression in primary human epidermal keratinocytes induces several MC lineage markers: Normal human epidermal keratinocytes (NHEK) were infected with a lentiviral vector coding for GLI1 and puromycin resistance. Following antibiotic selection, cells were harvested after 14 days of cultivation. (<b>A</b>) Immunoblot analysis was performed to confirm GLI1 expression (insert), and isolated RNA was subjected to complementary DNA (cDNA) synthesis and real-time PCR. Relative messenger RNA (mRNA) expression levels of the indicated Merkel cell lineage markers are given as mean (+ standard error of the mean (SEM)) of four independent experiments (* <span class="html-italic">p</span> value &lt; 0.05, paired <span class="html-italic">t</span> test) (mean CT value of the controls was used as reference). (<b>B</b>) Expression of GLI1, the MC progenitor (KRT17, SOX9) and the MC markers (SOX2, KRT8, and KRT20) was assessed by immunohistochemistry and relative protein expression quantification was performed on at least 1000 cells/condition using ImageJ software. Results are displayed as box and whiskers diagram with median, Q1, and Q3, as well as first and 99th percentile. These results were confirmed by two additional independent experiments (immunostaining and immunoblot) as shown in <a href="#app1-cancers-12-01989" class="html-app">Figure S3</a>. Uncropped membranes and Western blot signal quantifications are available in <a href="#app1-cancers-12-01989" class="html-app">Figures S8 and S9</a>, respectively.</p>
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<p>T antigens induce expression of some early MC differentiation markers in primary human keratinocytes. <b>A:</b> NHEKs were infected with a lentiviral vector coding for small T (sT) and truncated Large T (LT) as well as a puromycin resistance. Following antibiotic selection, cells were analyzed after 14 days of cultivation. (<b>A</b>) Immunoblot analysis confirmed LT expression, and microscopic inspection revealed a less-flattened phenotype and cultures reaching much higher densities. Under microscopic examination such cells harbored reduced cytoplasmic size compared to the controls, as confirmed using imageJ software (bar = 100 µm) (* <span class="html-italic">p</span> value &lt; 0.05, Mann–Whitney U test, <span class="html-italic">n</span> = 3 independent experiments). (<b>B</b>) Relative mRNA levels of the indicated Merkel cell differentiation markers (* <span class="html-italic">p</span> value &lt; 0.05, paired <span class="html-italic">t</span> test, <span class="html-italic">n</span> = 4 independent experiments), (<b>C</b>) Immunoblot demonstrated T antigens (TA)-induced KRT8 protein expression and immunohistochemistry additionally revealed KRT8 expression is restricted to a subpopulation of small- to medium-sized round cells. Furthermore, occasionally “dot like” staining was observed (white arrows). (<b>D</b>) Immunohistochemical assessment of the indicated MC markers in TA-expressing NHEK, control NHEK and the MCC cell line WaGa (bar = 100 µm). KRT8 induction by T antigens was confirmed in two additional independent experiments, which are depicted in <a href="#app1-cancers-12-01989" class="html-app">Figure S4</a>. For relative quantification of protein expression levels, at least 1000 cells/condition were evaluated using ImageJ software. Results are displayed as box and whiskers diagram with median, Q1, and Q3 as well as first and 99th percentile. Uncropped membranes and Western blot signal quantifications are available in <a href="#app1-cancers-12-01989" class="html-app">Figures S8 and S9</a>, respectively.</p>
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<p>Induction of late MC markers by combined expression of GLI1 and Merkel cell Polyomavirus (MCPyV) T antigens (TA) in primary keratinocytes. NHEKs were infected with a bicistronic lentiviral vector coding for GLI1 as well as sT and truncated LT. Under control of a second promoter, a pure resistance was expressed. Following antibiotic selection, cells were analyzed after 14 days of cultivation. (<b>A</b>) GLI1/TA combined ectopic expression was associated with formation of floating clusters of living cells in normal human epidermal keratinocytes (NHEK), while these findings were not observed in controls or when GLI1 and TA were transduced independently (PC: Phase contrast) (<a href="#app1-cancers-12-01989" class="html-app">Figure S4</a>) (<span class="html-italic">n</span> = 3 independent experiments). White arrows indicate the floating cells. (<b>B</b>,<b>C</b>) Immunohistochemical assessment of Merkel cell markers (SOX2, KRT8, and KRT20) expression levels in GLI1/T antigen-expressing NHEKs and controls. Immunohistochemistry was performed on the respective cells spotted on slides (2 × 10<sup>5</sup> cells/condition). B. Count of cells expressing the Merkel cell markers in GLI1/T antigens (TA)-expressing NHEK and controls (results are mean ±SEM of three independent experiments). Counting of positive cells was preferred to relative protein level quantification due to the low number of GLI1/TA-expressing cells. C. Representative photos of LT, GLI1, SOX2, KRT8, KRT18, and KRT20 expression in NHEK (controls), GLI1/TA-expressing NHEK, and the WaGa MCC cell line. White arrows indicate cells expressing the respective proteins. The results for two additional independent experiments are shown in <a href="#app1-cancers-12-01989" class="html-app">Figure S5</a>.</p>
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<p>MCPyV T antigens increase the half-life of ATOH1 (<b>A</b>) <b>Hemagglutinin</b> (HA)-tagged ATOH1- and/or TA-encoding plasmids were transfected either individually or combined into U2OS cells. After two days, real-time PCR and immunoblot analyses were performed. While <span class="html-italic">ATOH1</span> mRNA was not affected (mean ± SEM of three independent experiments), ATOH1 protein accumulation in the presence of TA was observed. (<b>B</b>) Co-transfection of a constant amount (0.3 µg) of HA-tagged ATOH1 and increasing amounts of TA in U2OS cells followed by immunoblot analysis. ATOH1-HA signals relative to actin were quantified using ImageJ. Mean ± SEM of three independent experiments was displayed. (<b>C</b>) Evaluation of ATOH1 half-life in absence or presence of T antigens. Twenty-four hours after transfection, HEK293 cells were exposed to the translation inhibitor cycloheximide (CHX) for variable durations (0–6 h). ATOH1-HA expression was then evaluated by immunoblot analysis and quantified using the Image J Software (mean ± SEM of three independent experiments are depicted). (<b>D</b>) A mutant of ATOH1-HA, in which the three serines at positions 331, 337, and 342 were all exchanged to alanines (ATOH1-HA-3A), was generated, and the impact of co-transfected TA on ATOH1-HA wild type and ATOH1-HA-3A expression was analyzed in CHX chase experiments (see C). Quantified signals relative to actin are given in the graphs below. (<b>E</b>) Co-transfection of ATOH1-HA-3A with increasing amounts of TA did not affect ATOH1 protein expression level (this was confirmed in a second independent experiment). Uncropped membranes and Western blot signal quantifications are available in <a href="#app1-cancers-12-01989" class="html-app">Figures S8 and S9</a>, respectively.</p>
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15 pages, 4929 KiB  
Article
Canine Epithelial Skin Tumours: Expression of the Stem Cell Markers Lgr5, Lgr6 and Sox9 in Light of New Cancer Stem Cell Theories
by Laura Bongiovanni, Chiara Brachelente, Eva Moreno and Monika M. Welle
Vet. Sci. 2020, 7(2), 62; https://doi.org/10.3390/vetsci7020062 - 8 May 2020
Cited by 5 | Viewed by 4701
Abstract
Evidence is accumulating that tumour development is driven by cancer stem cells (CSCs). In order to understand the presence and potential contribution of stem cells (SCs) as tumour-initiating cells in canine cutaneous tumours, we selected three putative SC markers (Lgr5, Lgr6 and Sox9) [...] Read more.
Evidence is accumulating that tumour development is driven by cancer stem cells (CSCs). In order to understand the presence and potential contribution of stem cells (SCs) as tumour-initiating cells in canine cutaneous tumours, we selected three putative SC markers (Lgr5, Lgr6 and Sox9) and investigated their expression pattern, level of protein and mRNA expression, in 43 canine hair follicle (HF) and 18 canine cutaneous epidermal tumours by immunohistochemistry and qRT-PCR, using normal skin samples as controls. Lgr5 protein expression was not detected in epidermal and HF tumours; however, Lgr5 mRNA overexpression was evident in some HF tumours. Sox9 was expressed in several tumour cases, both at the protein and mRNA level. The Lgr6 antibody tested was not suitable for formalin-fixed paraffin-embedded tissue samples, but Lgr6 gene showed higher expression in several samples of both HF and epidermal tumours compared with normal skin. Significantly higher mRNA expression levels of the three SC markers were found in trichoblastomas (TB) compared with basal cell carcinomas (BCC). The present results indicated that canine HF and epidermal tumours might have common tumour-initiating cells. The mRNA expression of the three selected SC markers, especially Lgr5, could be potentially useful in the distinction between canine TB and BCC. Full article
(This article belongs to the Section Anatomy, Histology and Pathology)
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Graphical abstract

Graphical abstract
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<p>The localization of Lgr5 was confirmed in specific regions of canine hair follicle in healthy skin. Lgr5 protein expression was immunohistochemically evident in the secondary germ of the telogen and early anagen hair follicle (HF). Magnification 40×.</p>
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<p>The localization of Sox9 was confirmed in specific regions of canine hair follicle in healthy skin. Sox9-positive cells in the innermost cell (IMC) layer of the outer root sheath (ORS) in the isthmus of late anagen hair follicles. Magnification 20×.</p>
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<p><span class="html-italic">Lgr5</span> and <span class="html-italic">Sox</span>9 are downregulated in epidermal tumours compared with normal skin. mRNA expression of <span class="html-italic">Sox9</span>, <span class="html-italic">Lgr5</span> and <span class="html-italic">Lgr6</span> in normal skin and epidermal tumours. Bars represent mean ± SEM. ** <span class="html-italic">p</span> &lt; 0.05; *** <span class="html-italic">p</span> &lt; 0.001. Normal skin tissues (normal skin, <span class="html-italic">n</span> = 3), basal cell carcinomas (BCC, <span class="html-italic">n</span> = 6), squamous cell carcinomas (SCC, <span class="html-italic">n</span> = 9).</p>
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<p>In SCC, Sox9-positive cells were mainly present in neoplastic cells showing basal cell morphology. Magnification 40×.</p>
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<p>In basosquamous SCC, high Sox9 expression was observed in tumour cells with basal cell morphology, mainly in the inner parts of the neoplastic islands. Magnification 20×.</p>
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<p>In BCC, a patchy distribution of immunostaining was observed. Magnification 40×.</p>
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<p>TB samples show the highest stem cell marker mRNA expression levels. mRNA expression of <span class="html-italic">Sox9, Lgr5</span> and <span class="html-italic">Lgr6</span> in normal skin and HF tumours. Bars represent mean ± SEM. No statistically significant differences were observed comparing each tumour with normal skin. Normal skin tissues (normal skin, <span class="html-italic">n</span> = 3), tricholemmomas (TL, <span class="html-italic">n</span> = 3), infundibular keratinizing acanthomas (IKA, <span class="html-italic">n</span> = 9), trichoblastomas (TB, <span class="html-italic">n</span> = 8), trichoepitheliomas (TE, <span class="html-italic">n</span> = 11), pilomatricomas (PM, <span class="html-italic">n</span> = 6).</p>
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<p>In IKA, Sox9-positive cells mainly present among the neoplastic cells of the basal cell layer of the tumour wall. Magnification 20×.</p>
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<p>TB, ribbon type: positive neoplastic cells in the inner parts of the tumour nests. Magnification 40×.</p>
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<p>TL, inferior type: scattered, positive nuclei evident in the neoplastic islands. Magnification 40×.</p>
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<p>PM: negative neoplastic cells with matrical cell morphology associated with single or small clusters of positive cells. Magnification 40×.</p>
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<p>TE: groups of positive neoplastic cells within the parts of the tumour with basal cell morphology. Negative cells with squamous differentiation. Magnification 40×.</p>
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<p><span class="html-italic">Sox9</span> and <span class="html-italic">Lgr6</span> mRNA are potential markers of malignancy in TE tumours. mRNA expression of <span class="html-italic">Sox9, Lgr5</span> and <span class="html-italic">Lgr6</span> in TE cases. When the average values of benign and malignant cases were compared within the TE group, differences were present for all the three markers. Two-tailed t-test: <span class="html-italic">Sox9</span>, not significant (n.s.) (<span class="html-italic">p</span> = 0.379); <span class="html-italic">Lgr5</span>, n.s. (<span class="html-italic">p</span> = 0.269); <span class="html-italic">Lgr6</span>, n.s. (<span class="html-italic">p</span> = 0.150).</p>
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