International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Automated Computational Detection, Quantitation, and Mapping of Mitosis in Whole-Slide Images for Clinically Actionable Surgical Pathology Decision Support.

BACKGROUND: Determining mitotic index by counting mitotic figures (MFs) microscopically from tumor areas with most abundant MF (hotspots [HS]) produces a prognostically useful tumor grading biomarker. However, interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling MF, coupled with computer-automated counting by image analysis, can improve reproducibility. A computational system for obtaining MF values across digitized whole-slide images (WSIs) was sought that would minimize impact of artifacts, generate values clinically relatable to counting ten high-power microscopic fields of view typical in conventional microscopy, and that would reproducibly map HS topography. MATERIALS AND METHODS: Relatively low-resolution WSI scans (0.50 µm/pixel) were imported in grid-tile format for feature-based MF segmentation, from naturally occurring canine melanomas providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells. Computer vision image processing was established to subtract key artifacts, obtain MF counts, and employ rotationally invariant feature extraction to map MF topography. RESULTS: The automated topometric HS (TMHS) algorithm identified mitotic HS and mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS topographically. Influence of dye, pigment, and extraneous structure artifacts was minimized. TMHS diagnostic decision support included image overlay graphics of HS topography, as well as a spreadsheet and plot of tile-based MF count values. TMHS performance was validated examining both mitotic HS counting and mapping functions. Significantly correlated TMHS MF mapping and metrics were demonstrated using repeat analysis with WSI in different orientation (R 2 = 0.9916) and by agreement with a pathologist (R 2 = 0.8605) as well as through assessment of counting function using an independently tuned object counting algorithm (OCA) (R 2 = 0.9482). Limits of agreement analysis support method interchangeability. MF counts obtained led to accurate patient survival prediction in all (n = 30) except one case. By contrast, more variable performance was documented when several pathologists examined similar cases using microscopy (pair-wise correlations, rho range = 0.7597-0.9286). CONCLUSIONS: Automated TMHS MF segmentation and feature engineering performance were interchangeable with both observer and OCA in digital mode. Moreover, enhanced HS location accuracy and superior method reproducibility were achieved using the automated TMHS algorithm compared to the current practice employing clinical microscopy.

Agreement in Histological Assessment of Mitotic Activity Between Microscopy and Digital Whole Slide Images Informs Conversion for Clinical Diagnosis.

Validating digital pathology as substitute for conventional microscopy in diagnosis remains a priority to assure effectiveness. Intermodality concordance studies typically focus on achieving the same diagnosis by digital display of whole slide images and conventional microscopy. Assessment of discrete histological features in whole slide images, such as mitotic figures, has not been thoroughly evaluated in diagnostic practice. To further gauge the interchangeability of conventional microscopy with digital display for primary diagnosis, 12 pathologists examined 113 canine naturally occurring mucosal melanomas exhibiting a wide range of mitotic activity. Design reflected diverse diagnostic settings and investigated independent location, interpretation, and enumeration of mitotic figures. Intermodality agreement was assessed employing conventional microscopy (CM40×), and whole slide image specimens scanned at 20× (WSI20×) and at 40× (WSI40×) objective magnifications. An aggregate 1647 mitotic figure count observations were available from conventional microscopy and whole slide images for comparison. The intraobserver concordance rate of paired observations was 0.785 to 0.801; interobserver rate was 0.784 to 0.794. Correlation coefficients between the 2 digital modes, and as compared to conventional microscopy, were similar and suggest noninferiority among modalities, including whole slide image acquired at lower 20× resolution. As mitotic figure counts serve for prognostic grading of several tumor types, including melanoma, 6 of 8 pathologists retrospectively predicted survival prognosis using whole slide images, compared to 9 of 10 by conventional microscopy, a first evaluation of whole slide image for mitotic figure prognostic grading. This study demonstrated agreement of replicate reads obtained across conventional microscopy and whole slide images. Hence, quantifying mitotic figures served as surrogate histological feature with which to further credential the interchangeability of whole slide images for primary diagnosis.

Expression of cyclooxygenase-2 in canine nasal carcinomas.

Cyclooxygenase(COX)-2 expression was evaluated in 24 paraffin-embedded canine nasal carcinoma tissue samples by immunohistochemistry. Several different tumor types were represented, including carcinomas, adenocarcinomas and squamous cell carcinomas. COX-2 expression was identified in 17/24 cases (71%). The proportion of positive cells expressing COX-2 ranged from 10 to 95% and COX-2 expression was predominantly localized in the cytoplasm. Treatment with a COX-2 inhibitor should be investigated, along with the utilization of COX-2 expression as a prognostic marker.

Multicenter case-control study of risk factors associated with development of vaccine-associated sarcomas in cats.

OBJECTIVE: To determine whether particular vaccine brands, other injectable medications, customary vaccination practices, or various host factors were associated with the formation of vaccine-associated sarcomas in cats. DESIGN: Prospective multicenter case-control study. ANIMALS: Cats in the United States and Canada with soft tissue sarcomas or basal cell tumors. PROCEDURE: Veterinarians submitting biopsy specimens from cats with a confirmed diagnosis of soft tissue sarcoma or basal cell tumor were contacted for patient medical history. Time window statistical analyses were used in conjunction with various assumptions about case definitions. RESULTS: No single vaccine brand or manufacturer within antigen class was found to be associated with sarcoma formation. Factors related to vaccine administration were also not associated with sarcoma development, with the possible exception of vaccine temperature prior to injection. Two injectable medications (long-acting penicillin and methyl prednisolone acetate) were administered to case cats more frequently than to control cats. CONCLUSIONS AND CLINICAL RELEVANCE: Findings do not support the hypotheses that specific brands or types of vaccine within antigen class, vaccine practices such as reuse of syringes, concomitant viral infection, history of trauma, or residence either increase or decrease the risk of vaccine-associated sarcoma formation in cats. There was evidence to suggest that certain long-acting injectable medications may also be associated with sarcoma formation.

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma.

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.