Computational Molecular Pathology Research for Cancer Diagnostics and Biomarkers

研究的目的是描述和开发人类疾病的新动物模型，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。其他目标包括开发用于评估和应用疾病生物标志物的新研究技术。在开发癌症诊断方法以及开发和描述人类癌症新模型的研究资源方面取得了进展。该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自动形态图像分析方法，以及粘膜黑色素瘤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生物标本组织切片中的组织学肿瘤和非肿瘤区域。开发和应用包括计算实验模型中生物标志物表达程度的算法。使用计算机辅助诊断的新方法进行了临床验证研究。验证数字病理学作为诊断中传统显微镜的替代品仍然是确保有效性的优先事项。多模态一致性研究通常侧重于通过全载玻片图像的数字显示和常规显微镜检查实现相同的诊断。在诊断实践中，尚未对整个切片图像中的离散组织学特征（如有丝分裂像）进行全面评估。为了进一步衡量传统显微镜与数字显示的初步诊断，12病理学家检查了113犬自然发生的粘膜黑色素瘤表现出广泛的有丝分裂活动的可重复性。设计反映了不同的诊断设置，并调查了独立的位置，解释和计数的有丝分裂像。采用常规显微镜CM 40和在20倍WSI 20和40倍WSI 40物镜放大率下扫描的全载玻片图像样本评估模态间一致性。从常规显微镜和全载玻片图像中获得了总计1647个有丝分裂像计数观察结果，以进行比较。配对观察的观察者内一致率为0.785至0.801，观察者间一致率为0.784至0.794。与传统显微镜相比，2种数字模式之间的相关系数相似，表明模式之间的非劣效性，包括以较低的20倍分辨率采集的整个载玻片图像。由于有丝分裂像计数用于几种肿瘤类型（包括黑色素瘤）的预后分级，8名病理学家中有6名使用全载玻片图像回顾性预测生存预后，相比之下，10名病理学家中有9名使用常规显微镜，首次评估全载玻片图像用于有丝分裂像预后分级。本研究证明了在常规显微镜和整个载玻片图像中获得的重复读数的一致性。因此，定量有丝分裂像作为替代组织学特征，进一步证明整个载玻片图像的可重复性，用于初步诊断。黑色素瘤治疗的新模型正在开发中。黑色素瘤是人类和犬的一种重要恶性肿瘤。与基因工程模型不同，散发性自然发生的犬黑色素细胞肿瘤与人类疾病有几个共同特征，这可能使犬研究成为更相关的临床前模型。犬黑色素瘤很少发生在阳光照射的部位，大多数自发发生在口腔。与人类一样，犬黑素细胞瘤的自然发生谱包括与痣类似的良性病变，以及侵袭性原发性黑色素瘤和广泛转移。与人类一样，宠物犬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改变、细胞来源、流行病学、与紫外线辐射的关系以及从良性肿瘤到恶性肿瘤的进展方面存在差异。与人类皮肤黑色素瘤相比，犬和人类粘膜黑色素瘤（MM）似乎罕见地含有BRAF，NRAS和c-kit突变，尽管两个物种都具有AKT和MAPK信号转导激活。我们的结论是，自然发生的犬和人MM的临床和组织病理学特征有显着的重叠。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。评价的大多数人和犬MM均显示RAS/ERK和/或PI 3 K/mTOR信号通路激活。具有反映犬中天然存在差异的不同ERK和AKT/mTOR活化水平的犬MM细胞系对MEK抑制和双重PI 3 K/mTOR抑制敏感。研究的重点是继续确定联合疗法和递送策略，以对抗肿瘤生长和转移。这些发现继续确立了当多种介质在具有Ras/ERK和PI 3 K/mTOR通路活化的黑色素瘤中同时靶向时的协同治疗功效的证据。

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.