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BackgroundCanine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA-1, ionised calcium-binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features.ObjectivesThis study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single-molecule RNA visualisation in formalin-fixed paraffin-embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS.MethodsReverse transcription polymerase chain reaction (RT-PCR) and western blot analysis for IBA-1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA-ISH for IBA-1 and CD204 was performed on the DH82 cell line to validate the RNA-ISH probes. RNA-ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA-1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls.ResultsRNA-ISH and IHC showed moderate to strong expression for IBA-1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA-ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration.ConclusionRNA-ISH for CD204 and IBA-1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.

The count of mitotic figures (MFs) observed in hematoxylin and eosin (H&E)-stained slides is an important prognostic marker, as it is a measure for tumor cell proliferation. However, the identification of MFs has a known low inter-rater agreement. In a computer-aided setting, deep learning algorithms can help to mitigate this, but they require large amounts of annotated data for training and validation. Furthermore, label noise introduced during the annotation process may impede the algorithms' performance. Unlike H&E, where identification of MFs is based mainly on morphological features, the mitosis-specific antibody phospho-histone H3 (PHH3) specifically highlights MFs. Counting MFs on slides stained against PHH3 leads to higher agreement among raters and has therefore recently been used as a ground truth for the annotation of MFs in H&E. However, as PHH3 facilitates the recognition of cells indistinguishable from H&E staining alone, the use of this ground truth could potentially introduce an interpretation shift and even label noise into the H&E-related dataset, impacting model performance. This study analyzes the impact of PHH3-assisted MF annotation on inter-rater reliability and object level agreement through an extensive multi-rater experiment. Subsequently, MF detectors, including a novel dual-stain detector, were evaluated on the resulting datasets to investigate the influence of PHH3-assisted labeling on the models' performance. We found that the annotators' object-level agreement significantly increased when using PHH3-assisted labeling (F1: 0.53 to 0.74). However, this enhancement in label consistency did not translate to improved performance for H&E-based detectors, neither during the training phase nor the evaluation phase. Conversely, the dual-stain detector was able to benefit from the higher consistency. This reveals an information mismatch between the H&E and PHH3-stained images as the cause of this effect, which renders PHH3-assisted annotations not well-aligned for use with H&E-based detectors. Based on our findings, we propose an improved PHH3-assisted labeling procedure. [ABSTRACT FROM AUTHOR]

Simple Summary: We investigated a new method for diagnosing and predicting outcomes in canine pulmonary carcinoma. We developed a deep learning-based algorithm that accurately detects tumor nuclei and subsequently measures size and shape parameters. The variation in nuclear size and shape (nuclear pleomorphism) is a crucial malignancy criterion used in the current grading system for canine pulmonary carcinoma. Pathologists currently evaluate it and classify it according to a three-tier system. Manual morphometry is a more objective approach where tumor nuclei are individually encircled and analyzed. This task can be easily performed by an algorithm. Our algorithm's accuracy in correctly detecting and segmenting tumor nuclei was considered good when compared to manual morphometry. By comparing automated morphometry with conventional prognostic tests, such as pathologists' estimates, mitotic count, histological grading, and clinical staging, we found that our approach was equally accurate in terms of prognostic value. The algorithm's advantage lies in its high reproducibility and efficiency. Automated evaluation of nuclear pleomorphism can enhance the efficiency and reliability of canine pulmonary carcinoma diagnosis and grading, effectively addressing issues of inter-observer reproducibility. However, further optimization of the algorithm and validation with a larger study group is necessary to confirm our findings. The integration of deep learning-based tools into diagnostic workflows is increasingly prevalent due to their efficiency and reproducibility in various settings. We investigated the utility of automated nuclear morphometry for assessing nuclear pleomorphism (NP), a criterion of malignancy in the current grading system in canine pulmonary carcinoma (cPC), and its prognostic implications. We developed a deep learning-based algorithm for evaluating NP (variation in size, i.e., anisokaryosis and/or shape) using a segmentation model. Its performance was evaluated on 46 cPC cases with comprehensive follow-up data regarding its accuracy in nuclear segmentation and its prognostic ability. Its assessment of NP was compared to manual morphometry and established prognostic tests (pathologists' NP estimates (n = 11), mitotic count, histological grading, and TNM-stage). The standard deviation (SD) of the nuclear area, indicative of anisokaryosis, exhibited good discriminatory ability for tumor-specific survival, with an area under the curve (AUC) of 0.80 and a hazard ratio (HR) of 3.38. The algorithm achieved values comparable to manual morphometry. In contrast, the pathologists' estimates of anisokaryosis resulted in HR values ranging from 0.86 to 34.8, with slight inter-observer reproducibility (k = 0.204). Other conventional tests had no significant prognostic value in our study cohort. Fully automated morphometry promises a time-efficient and reproducible assessment of NP with a high prognostic value. Further refinement of the algorithm, particularly to address undersegmentation, and application to a larger study population are required. [ABSTRACT FROM AUTHOR]

Telangiectatic osteosarcoma is a rare variant of osteosarcoma histologically and clinically similar to hemangiosarcoma (HSA). This case series describes the imaging and cytologic features of four histologically confirmed telangiectatic osteosarcomas, including the use of cytochemical stains. Alkaline phosphatase (ALP) was applied to Wright–Giemsa‐stained cytology slides, and Factor VIII immunohistochemistry was evaluated. Cytologic characteristics included atypical mesenchymal cells with evidence of acute and chronic hemorrhage. Telangiectatic osteosarcoma cases had positive ALP cytochemical staining, while control HSA cases were negative. Factor VIII immunohistochemistry was negative in telangiectatic osteosarcoma and positive in HSA. Cytologic diagnosis of telangiectatic osteosarcoma with positive ALP cytochemical staining can help differentiate this neoplasm from HSA. [ABSTRACT FROM AUTHOR]

The Veterinary Cancer Guidelines and Protocols (VCGP) group has developed guidelines for developing, reporting, and validating histologic tumor grading systems. These guidelines aim to standardize tumor evaluation in order to improve the care of animals with cancer. The grading systems should be reproducible, relevant to prognosis, and realistic in terms of applicability in a routine diagnostic setting. The VCGP group encourages collaboration from various disciplines and emphasizes the importance of standardized clinical outcomes and statistical assessment. The guidelines also suggest comparing histologic grade with other assays and encourage the publication of grading systems in peer-reviewed journals with sufficient details for replication and validation. Validation studies should be conducted before implementing the grading system on clinical cases. [Extracted from the article]

Fungi in the genus Aspergillus are some of the most common fungal pathogens in veterinary species, primarily affecting the respiratory tract. In both human and veterinary cases, calcium oxalate crystals have been documented in sites of Aspergillus infection. Cases in multiple species (16 birds, 15 horses, 5 dogs, 1 ox, and 1 dolphin) were identified that had either positive cultures for Aspergillus sp., or had conidiophores present that could be identified as belonging to the genus Aspergillus. Histologic slides were examined to confirm the presence of oxalate crystals and how often they were identified on the original report. Calcium oxalate deposition was detected in 14 of 38 cases examined, including A. fumigatus, A. versicolor, A. niger, and unspecified Aspergillus sp. infections. Calcium oxalate crystals were identified in 11 of 16 avian cases, as well as in 1 of 1 bovine, 1 of 15 equine, and 1 of 5 canine cases. Crystals were described in only 3 of the 14 original pathology reports of these cases, indicating that identification and reporting of crystals in histologic specimens could be improved. All the tissues with crystals were respiratory tissues with air interfaces, including nasal sinus, trachea, syrinx, lung, and air sac. In cases with crystals identified on H&E-stained sections, crystals were frequently not present or were fewer in number in tissue sections stained with Gomori methenamine silver and periodic acid–Schiff. Routine polarization of slides of fungal infections, especially in the respiratory tract, should be considered to check for calcium oxalate crystals. [ABSTRACT FROM AUTHOR]

For the development of an effective HIV-1 vaccine, evolutionarily conserved epitopes between feline and human immunodeficiency viruses (FIV and HIV-1) were determined by analyzing overlapping peptides from retroviral genomes that induced both anti-FIV/HIV T cell-immunity in the peripheral blood mononuclear cells from the FIV-vaccinated cats and the HIV-infected humans. The conserved T-cell epitopes on p24 and reverse transcriptase were selected based on their robust FIV/HIV-specific CD8+ cytotoxic T lymphocyte (CTL), CD4+ CTL, and polyfunctional T-cell activities. Four such evolutionarily conserved epitopes were formulated into four multiple antigen peptides (MAPs), mixed with an adjuvant, to be tested as FIV vaccine in cats. The immunogenicity and protective efficacy were evaluated against a pathogenic FIV. More MAP/peptide-specific CD4+ than CD8+ T-cell responses were initially observed. By post-third vaccination, half of the MAP/peptide-specific CD8+ T-cell responses were higher or equivalent to those of CD4+ T-cell responses. Upon challenge, 15/19 (78.9%) vaccinated cats were protected, whereas 6/16 (37.5%) control cats remained uninfected, resulting in a protection rate of 66.3% preventable fraction (p = 0.0180). Thus, the selection method used to identify the protective FIV peptides should be useful in identifying protective HIV-1 peptides needed for a highly protective HIV-1 vaccine in humans. [ABSTRACT FROM AUTHOR]