Mitotic count (MC) is an important element for grading canine cutaneous mast cell tumors (ccMCTs) and is determined in ten consecutive high-power fields with the highest mitotic activity. However, there is variability in area selection between pathologists. In this study, the MC distribution and the effect of area selection on the MC were analyzed in ccMCTs. Two pathologists independently annotated all mitotic figures in whole-slide images of 28 ccMCTs (ground truth). Automated image analysis was used to examine the ground truth distribution of the MC throughout the tumor section area, which was compared with the manual MCs of 11 pathologists.Computerized analysis demonstrated high variability of the MC within different tumor areas. There were 6 MCTs with consistently low MCs (MC<7 in all tumor areas), 13 cases with mostly high MCs (MC ≥7 in ≥75% of 10-hpf-areas) and 9 borderline cases with variable MCs around 7, which is a cut-off value for ccMCT grading. There was inconsistency among pathologists in identifying the areas with the highest density of mitotic figures throughout the three ccMCT groups; only 51.9% of the counts were consistent with the highest 25% of the ground truth MC distribution. Regardless, there was substantial agreement between pathologists in detecting tumors with MC≥7.Falsely low MCs below 7 mainly occurred in 4/9 borderline cases that had very few ground truth areas with MC≥7. The findings of this study highlight the need to further standardize how to select the region of the tumor in which to determine the MC. Keywords: Area selection, high-power field, mitotic activity, mitotic figure distribution, tumor grading, tumor periphery. 3 Aubreville M, Bertram CA, Klopfleisch R, Maier A. Field of interest proposal for augmented mitotic cell count: Comparison of two convolutional networks.

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BackgroundBreast cancer is one of the leading causes of cancer deaths. Triple-negative breast cancer (TNBC), an immunophenotype defined by the absence of immunolabeling for estrogen receptor (ER), progesterone receptor (PR) and HER2 protein, has a highly aggressive behavior. A subpopulation of TNBCs exhibit a basal-like morphology with immunohistochemical positivity for cytokeratins 5/6 (CK5/6) and/or epidermal growth factor receptor (EGFR), and have a high incidence of BRCA (breast cancer susceptibility) mutations. Feline mammary adenocarcinomas (FMAs) are highly malignant and share a similar basal-like subtype. The purpose of this study was to classify FMAs according to the current human classification of breast cancer that includes evaluation of ER, PR and HER2 status and expression of basal CK 5/6 and EGFR. Furthermore, we selected triple negative, basal-like FMAs to screen for BRCA mutations similar to those described in human TNBC.MethodsTwenty four FMAs were classified according to the current human histologic breast cancer classification including immunohistochemistry (IHC) for ER, PR HER2, CK5/6 and EGFR. Genetic alteration and loss of heterozygosity of BRCA1 and BRCA2 genes were analyzed in triple negative, basal-like FMAs.ResultsIHC for ER, PR and HER2 identified 14 of the 24 (58%) FMAs as a triple negative. Furthermore, 11of these 14 (79%) triple negative FMAs had a basal-like subtype. However, no genetic abnormalities were detected in BRCA1 and BRCA2 by direct sequencing and loss of heterozygosity analysis.ConclusionFMAs are highly aggressive neoplasms that are commonly triple negative and exhibit a basal-like morphology. This is similar to human TNBC that are also commonly classified as a basal-like subtype. While sequencing of a select number of triple negative, basal-like FMAs and testing for loss of heterozygosity of BRCA1 and BRCA2 did not identify mutations similar to those described in human TNBC, further in-depth evaluation is required to elucidate a potential role of BRCA in the tumorigenesis of triple negative, basal-like FMAs. The strong similarities in clinical behavior, morphology and IHC phenotype suggest that triple negative, basal-like FMAs may be a suitable spontaneous animal model for studying novel therapeutic approaches against human basal-like TNBC.

Canine oral papillomavirus (CPV1, also known as COPV), the most common cause of non-neoplastic papillomas, has not been shown to cause squamous cell carcinomas (SCC). Furthermore, malignant transformation of benign papillomas to SCC has only been reported in a single group of dogs with severe combined immunodeficiency infected with CPV2. Here, we report a series of 7 dogs with benign CPV1-associated papillomas with histologic evidence of CPV1 causing malignant transformation to carcinoma in situ and ultimately SCC. Expression of p53 and p16 proteins in CPV1-infected cells within the benign papillomas and lesions that progressed into SCC also supported an association between papillomavirus and malignant transformation. Moreover, our retrospective analysis indicated that while there have been increased numbers of viral papillomas with malignant transformation, the number of annually diagnosed canine viral papillomas has remained constant over the past decade in our laboratory. We speculate that either an altered host immunity from increased usage of immunosuppressive drugs or changing environmental factors, e.g. increase exposure to UV radiation, may cause an increased oncogenic potential of this “low-risk” virus. This study aims to raise awareness of the malignant potential of CPV1 and to encourage further investigations into the cause of this suspected change in its oncogenic potential.

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer's patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct<15) in the affected small intestine, and immunohistochemistry detected large amounts of CPV-2 antigen in intestinal crypt epithelium and Kupffer cells but few positive macrophages in lymphoid organs. All puppies had marked sinusoidal histiocytosis and multifocal granulomatous inflammation in mesenteric lymph nodes and spleen, prompting additional RT-PCR testing for canine circovirus 1 (CaCV-1). Very high levels of CaCV-1 DNA (Ct<13) were detected in small intestine, lymph nodes, and spleen. In situ hybridization for CaCV-1 detected rare positive nuclei of regenerating crypt epithelium but abundant amounts of CaCV-1 nucleic acid in the cytoplasm and nuclei of histiocytes in all lymphoid tissues, including granulomatous inflammatory foci and hepatic Kupffer cells. Significant levels of CaCV-1 DNA were detected in blood and serum (Ct as low as 13) but not feces from 3 surviving dogs at 2 months or 1 year after the outbreak, respectively. We hypothesize that CPV-2 infection predisposed dogs to CaCV-1 infection and ultimately resulted in more severe clinical disease.