Research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize a model's relevance, addressing critical barriers to research progress. Additional aims include the development of new research technologies for the evaluation and application of disease biomarkers. Progress was made in developing cancer diagnostics and in research resources useful in developing and characterizing new models of human cancer. This research project included developing capabilities in molecular diagnostics for cancer models, developing methods for automated morphometric image analysis of cancer specimens for quantitative pathology, and preclinical development of targeted therapy for mucosal melanoma. Unique spatial-spectral image analysis algorithms were developed for applying automated pattern recognition morphometric image analysis to quantify histologic tumor and non-tumor areas in biospecimen tissue sections. Development and application included algorithms to compute the degree of differentiation in experimental tumors mechanistically differentiated by altering various oncogenic signals under transcription factor control. Conduct of a multi-center clinical diagnostic study in collaboration with the US Food and Drug Administration is leading to useful criteria for validation of whole slide digital pathology tissue image platforms for clinical cancer diagnosis. Further progress involved determining that mitotic index by counting mitotic figures (MF) microscopically from tumor areas with most abundant MF (hot spots, (HS)), produces a prognostically useful tumor grading biomarker. However, the process is tedious and interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling of MF, coupled with computer-automated counting by image analysis can improve reproducibility. A computational system for obtaining MF values across digitized whole slide images (WSI) was sought that would minimize common tissue and stain artifacts, generate values clinically relatable to counting ten high-power microscopic fields-of-view (FOV) typical in conventional microscopy, and that would reproducibly map mitotic HS topography. Relatively low-resolution WSI scans (0.47 pixel/um) were imported in a grid-tile format at 4X magnification for feature-based MF segmentation, from a naturally occurring spontaneous canine melanoma tumor model providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells detected with red chromogen. Potentially confounding artifacts were addressed using a series of computer vision processing filters. Image processing was established to obtain MF counts and employ rotationally-invariant feature extraction to map MF topography. The automated topometric hot spot (TMHS) algorithm developed mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS activity topographically. Variables due to presence of dye, pigment and extraneous structure artifacts were minimized. TMHS tumor grading diagnostic decision-support provided at case read-out included image overlay graphics of HS topography, a plot of image grid-tile MF counts, as well as a spreadsheet of tile-based MF count values, each corresponding to ten high-power FOV. TMHS performance was validated examining both mitotic HS mapping and counting functions in agreement analyses with a pathologist, as well as with MF counts acquired by an independently-tuned object counting algorithm. Repeat analysis of clinical specimens using the automated TMHS mapping algorithm provided significant evidence of method interchangeability. Enhanced HS location accuracy, compared to pathologist selection, was achieved using TMHS. Automated TMHS provides practical improvements for clinically-actionable mitotic HS topography and MF counting from WSI, the prognostic utility of which was significantly correlated with the standard of care. New model development is taking place for melanoma treatment. Melanoma represents a significant malignancy in humans and dogs. Distinct from genetically engineered models, sporadic naturally occurring canine melanocytic neoplasms share several characteristics with human disease that could make investigation in dogs a more relevant pre-clinical model. Canine melanomas rarely arise in sun-exposed site and most occur spontaneously in the oral cavity. The spectrum of naturally occurring canine melanocytic neoplasia, as is true in people, includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. As in humans, distinct melanoma subtypes 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 pet dogs. Both canine and human mucosal melanomas (MM) appear to harbor BRAF, NRAS and c-kit mutations uncommonly, compared to 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 naturally occurring canine and human MM. Naturally occurring canine oral cavity melanoma is being explored further as a pre-clinical model for human melanoma. The majority of human and canine MM evaluated exhibited RAS/ERK and/or PI3K/mTOR signaling pathway activation. Canine MM cell lines, with varying ERK and AKT/mTOR activation levels reflective of naturally occurring differences in dogs, were sensitive to MEK inhibition and dual PI3K/mTOR inhibition. Additional two-drug combinations synergistically decreased cell survival in association with annexin V expression, as well as altered expression of cell cycle regulatory proteins and Bcl-2 family proteins. In combination, the two drugs targeted their respective signaling pathways, potentiating reduction of pathway mediators p-ERK, pAKT, p-S6, and 4EBP1 in vitro, and in association with significantly inhibited solid tumor growth in MM xenografts in mice. These findings provide evidence of synergistic therapeutic efficacy when multiple mediators are simultaneously targeted in melanoma with Ras/ERK and PI3K/mTOR pathway activation.
Showing the most recent 10 out of 32 publications
