Combined colour deconvolution and artificial intelligence approach for region-selective immunohistochemical labelling quantification: The example of alpha smooth muscle actin in mouse kidney.

Immunohistochemical (IHC) localisation of protein expression is a widely used tool in pathology. This is semi-quantitative and exhibits substantial intra- and inter-observer variability. Digital approaches based on stain quantification applied to IHC are precise but still operator-dependent and time-consuming when regions of interest (ROIs) must be defined to quantify protein expression in a specific tissue area. This study aimed at developing an IHC quantification workflow that benefits from colour deconvolution for stain quantification and artificial intelligence for automatic ROI definition. The method was tested on 10 whole slide images (WSI) of alpha-smooth muscle actin (aSMA) stained mouse kidney sections. The task was to identify aSMA-positive areas within the glomeruli automatically. Total aSMA detection was performed using two channels (DAB, haematoxylin) colour deconvolution. Glomeruli segmentation within the same IHC WSI was performed by training a convolutional neural network with annotated examples of glomeruli. For both aSMA and glomeruli, binary masks were created. Co-localisation was performed by overlaying the masks and assigning red/green colours, with yellow indicative of a co-localised signal. The workflow described and exemplified using the case of aSMA expression in glomeruli can be applied to quantify the expression of IHC markers within different structures of immunohistochemically stained slides. The technique is objective, has a fully automated threshold approach (colour deconvolution phase) and uses AI to eliminate operator-dependent steps

Primary Corneal Squamous Cell Carcinoma in a Dog: Clinical and Histopathological Evaluation

An 8-year-old male pug with a 12-month history of a progressive nonpainful mass on the left cornea was evaluated. Ocular examination showed a severe bilateral keratoconjunctivitis sicca, pigmentary keratitis, and an exophytic irregular pink mass occupying approximately 75% of the total corneal surface of the left eye. A squamous cell carcinoma (SCC) was suspected on cytology, and clinical investigations showed no evidence of metastases. A transpalpebral enucleation was therefore performed, and the diagnosis of SCC was confirmed on histopathology. Immunohistochemical investigations showed that the neoplastic cells were pan-cytokeratin positive and vimentin negative. Additionally, nuclei immunoreactive to Ki-67 antigen were detected. Tumor cells were also negative to p53. Immunoreactivity to COX-2 was found in less than 10% of the neoplastic cells. No adjuvant therapies were instituted, and no evidence of local recurrence or distance metastasis was identified during the 24-month follow-up period

Co-localization of PTEN and E-cadherin in canine mammary hyperplasias and benign and malignant mammary tumours

Fifty-four canine mammary lesions (15 hyperplasias, 7 adenomas and 32 carcinomas) were submitted to immunohistochemical analysis for the evaluation of PTEN and E-cadherin co-expression. Subjects bearing mammary carcinomas were also submitted to a 2-year follow-up study to compare immunohistochemical results with overall survival All the hyperplastic samples stained positive for both markers, 100% of adenomas were positive for PTEN and 86% for E-cadherin, and 69% and 34% of carcinomas were positive for PTEN and E-cadherin, respectively. Statistical analysis showed a positive correlation between these two proteins both considering all (p <0.01) or malignant tumours (p <0.05). The female dogs bearing tumours positively-stained for both markers had a longer overall survival (p <0.05) and absence of lymphatics invasion (p <0.05). Simultaneous double immunofluorescence confirmed the co-localization of the two proteins in neoplastic cells. Results reported in this study confirm the tumor suppressor effect of these two molecules

Expression Analysis of MicroRNAs in FFPE samples of canine cutaneous and oral melanoma by RT-qPCR

MicroRNA (miRNA), a class of small, non-coding RNA - regulating post-transcriptionally protein expression - are emerging as clinical biomarkers in many pathologies, including cancer [1]. Since miRNA are supposed to represent fundamental key regulators, better understanding of melanoma biology is essential to improve staging and therapy. The aim of the study was to investigate whether miRNA expression can vary in canine melanoma samples derived from formalin-fixed-paraffin-embedded (FFPE) tissues. Experimental design of the study included three groups, each one composed of 7 animals: i) control healthy skin group ii) oral melanoma group iii) skin melanoma group. The histhopatology and immunoistochemistry details of dogs included in the study are previously reported  [2]. Two tissue slides were used for miRNA extraction. The expression levels of seven miRNA - miR-145, miR-146a, miR-425-5p, miR-223, miR-365, miR-155 and miR-134 - were detected and assessed by qPCR using TaqMan® probes [3-5]. Five miRNA were significantly up-(n=3) or down-(n=2) regulated. In details, miR-146a and miR-155 abundance was increased as compared with control in both oral and skin melanoma (Fig 1 B,E) (p = 0.004 and 0.014 and p = 0.043 and 0.035 respectively), while the levels of miR-145 and miR-365 were lower (Fig 1 A,D) (p = 0.018 and 0.008 and p = 0.01 and 0.028, respectively). MiR-425-5p was upregulated (p = 0.039) only in skin melanoma (Fig. 1 C). Furthermore, functional analysis, carried out using miRNet web-based tool, showed that 76 genes related to cancer-associated pathways were possible target of these five microRNA (p = 6.95E-9); in particular, 21 target genes were associated with melanoma (p = 1.47E-5), including BRAF and CDK, E2F, FGF and PIK3 families. In conclusion, miR-145, miR-146a, miR-425-5p, miR-365 and miR-155 are differentially expressed in melanoma and healthy FFPE samples, suggesting that they may play a role in canine melanoma pathogenesis and/or progression

Correlation between Tumour Associated Macrophage (TAM) Infiltration and Mitotic Activity in Canine Soft Tissue Sarcomas†

Tumour-associated macrophages (TAMs) are an important part of the tumour microenvironment but knowledge of their distribution in canine soft tissue sarcomas (STSs) is limited to absent. We analysed 38 STSs retrieved from the veterinary pathology archive; oral and visceral STSs, synovial cell sarcoma, tumours of histiocytic origin, haemangiosarcoma, carcinosarcomas, and undifferentiated tumours were excluded. Iba-1 positive, non-neoplastic tumour infiltrating cells (morphologically indicative of macrophages) were classified as TAMs and were counted in 10 consecutive tumours areas, where no necrosis or other inflammatory cells could be identified. Associations between numbers of TAMs and mitoses, differentiation, and necrosis scores or grade were investigated. TAMs were evident in all STSs and ranged between 6% to 62% of the cells in the microscopic field. The number of TAMs positively correlated with the STSs’ histologic grade. When the components of the grade were analysed separately, TAMs were statistically correlated with mitoses, but not with differentiation or necrosis score. The present findings suggest that TAMs are present in higher numbers when STS proliferation is the predominant feature that drives tumour grade. The abundant presence of TAMs in high-grade STSs may also increase the likelihood of a pathologist misdiagnosing STS for histiocytic sarcoma