Computational Molecular Pathology Research for Cancer Diagnostics and Biomarkers

进行研究是为了描述和开发新的人类疾病动物模型，并开发更好地描述模型相关性的方法，解决研究进展的关键障碍。其他目标包括为评估和应用疾病生物标记物开发新的研究技术。在开发癌症诊断学和研究资源方面取得了进展，这些资源有助于开发和表征新的人类癌症模型。这项研究项目包括开发癌症模型的分子诊断能力，开发用于定量病理学的癌症标本的自动形态图像分析方法，以及针对粘膜黑色素瘤的靶向治疗的临床前开发。开发了独特的空间光谱图像分析算法，用于应用自动模式识别形态测量图像分析来量化组织学组织切片中的肿瘤和非肿瘤区域。开发和应用包括在实验模型中计算生物标记物表达程度的算法。对使用计算机辅助诊断的新方法进行了临床验证研究。确认数字病理学在诊断中替代传统显微镜仍然是确保有效性的优先事项。多通道一致性研究通常侧重于通过数字显示整个切片图像和常规显微镜来实现相同的诊断。在诊断实践中，对整个切片图像中离散的组织学特征的评估，例如有丝分裂图形，还没有得到彻底的评估。为了进一步评估常规显微镜和数字显示在初步诊断中的互换性，12名病理学家检查了113只自然发生的狗粘膜黑色素瘤，这些黑色素瘤表现出广泛的有丝分裂活动。设计反映了不同的诊断环境，并调查了有丝分裂图形的独立定位、解释和计数。使用常规显微镜CM40和WSI20倍WSI20和40倍WSI40客观放大扫描整个幻灯片图像样本，评估不同模式之间的一致性。从常规显微镜和整个切片图像中可以获得总计1647个有丝分裂图形计数的观察结果，以供比较。配对观察的观察者内符合率为0.785~0.801，观察者间符合率为0.784~0.794。两种数字模式之间的相关系数，与传统显微镜相比，相似，提示不同模式之间不存在劣势，包括以较低20倍分辨率获取的整个幻灯片图像。随着有丝分裂像计数用于包括黑色素瘤在内的几种肿瘤类型的预后分级，8名病理学家中有6名使用全切片图像回顾预测生存预后，相比之下，常规显微镜下10名中有9名，这是对整个切片图像进行有丝分裂图像预后分级的首次评估。这项研究证明了通过常规显微镜和整个玻片图像获得的重复读数的一致性。因此，有丝分裂像的量化可以作为替代的组织学特征，进一步证明整个切片图像在初步诊断中的互换性。治疗黑色素瘤的新模式正在开发中。黑色素瘤是人类和狗的一种重要的恶性肿瘤。与基因工程模型不同，零星的自然发生的犬类黑素细胞肿瘤与人类疾病有几个共同的特征，这可能使对狗的研究成为更相关的临床前模型。犬类黑色素瘤很少发生在阳光暴晒的部位，大多数自发发生在口腔。自然发生的犬类黑素细胞瘤的谱系，在人类中是真实的，包括一些类似痣的良性病变，以及侵袭性的原发黑色素瘤，以及广泛的转移。与人类一样，宠物狗中也可能存在不同的黑色素瘤亚型，这些亚型在体细胞和易感生殖系遗传变化、细胞起源、流行病学、与紫外线辐射的关系以及从良性到恶性肿瘤的进展方面都存在差异。与人类皮肤黑色素瘤相比，犬和人粘膜黑色素瘤(MM)似乎都罕见地含有BRAF、NRAS和c-kit突变，尽管这两个物种都有AKT和MAPK信号激活。我们的结论是，自然发生的犬和人类MM的临床和组织病理学特征有显著的重叠。自然发生的犬口腔黑色素瘤正在进一步探索，作为人类黑色素瘤的临床前模型。大多数人和犬多发性骨髓瘤表现出RAS/ERK和/或PI3K/mTOR信号通路的激活。犬MM细胞系对MEK抑制和PI3K/mTOR双重抑制敏感，其ERK和AKT/mTOR活性水平的变化反映了犬自然存在的差异。研究的重点是继续确定联合疗法和给药策略，以对抗肿瘤的生长和转移。这些发现继续证明，当多种介质同时靶向RAS/ERK和PI3K/mTOR通路激活的黑色素瘤时，具有协同治疗效果。

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 extent of biomarker expression in experimental models. Clinical validation studies were conducted for new approaches using computer assisted diagnostics. 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 times WSI20 and at 40 times 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 times 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 images 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. 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. Research has focused on continuing to identify combination therapies and delivery strategies to combat tumor growth and metastasis. These findings continue to establish evidence of synergistic therapeutic efficacy when multiple mediators are simultaneously targeted in melanoma with Ras/ERK and PI3K/mTOR pathway activation.