Computational Molecular Pathology Research for Cancer Diagnostics and Biomarkers

开展研究的目的是表征和开发新的人类疾病动物模型，并开发更好地表征模型中产生的疾病相关性的手段，解决研究进展的关键障碍。其他目标包括开发新的研究技术和手段来询问癌症组织，以评估和应用疾病生物标志物。该研究还旨在将癌症治疗方法转化为临床应用。在开发有助于开发和确定人类癌症新模型和癌症诊断的研究资源方面取得了进展。该研究产生了用于检测和量化核膜中促进转移的蛋白质的方法和计算能力，有助于了解细胞感知和转移的机制。乳腺癌建模的工作重点是人类乳腺癌中CLIC4表达的升高及其与女性早期侵袭和不良预后的一致性。宿主微环境中的CLIC4是肺转移所必需的，而宿主CLIC4的缺失导致模型中原发肿瘤和转移前肺的微环境不利于肿瘤生存和肺定植。特别是，通过计算分子组织分析发现，缺乏CLIC4的小鼠的原发肿瘤微环境会发生坏死和血管异常。研究还集中在肿瘤免疫学中一个高度热门和突出的问题。在肺腺癌中，我们共同提出驱动癌基因是否影响浸润肺的免疫细胞种类或其激活状态。采用这一参考框架是为了其潜在的临床相关性。我们分析了几种肺腺癌的免疫细胞浸润情况，主要评估了巨噬细胞和T细胞亚群，以及这些肿瘤中的反应性肿瘤相关纤维增生。在一个小的，高度管理的一组样本中，所有的样本在癌细胞中都有一定程度的免疫反应浸润。使用一组有限的参数（CD8, CD68, CD4, FOXP3, PDL1），我们没有观察到肿瘤之间的明显差异，其中突变的EGFR是驱动癌基因，而突变的KRAS。然而，纤维化程度似乎与淋巴细胞浸润有关。这组分析有力地证明了肿瘤异质性背景下免疫细胞浸润的复杂性。最后，该研究提供了T调节性淋巴细胞的存在、CCL5的高表达和CD8+效应功能在人肺腺癌中的相关性。粘膜黑色素瘤联合靶向治疗的药物开发工作也在继续。人和犬粘膜黑色素瘤之间的临床和病理相关性是实质性的，狗的自发性自然发生的粘膜黑色素瘤的相对较高的发病率为预测建模提供了一个有希望的机会。与人类皮肤黑色素瘤相比，犬类和人类粘膜黑色素瘤似乎都不常见BRAF、NRAS和c-kit突变，尽管两者都具有AKT和MAPK信号激活。人类和犬粘膜黑色素瘤的基因组景观呈现高度多样化，通常缺乏与皮肤黑色素瘤相关的复发热点突变。尽管仍有很多有待确定，但有证据表明，Ras/MAPK和/或PI3K/AKT/mTOR信号通路激活在两种物种中都很常见，可能是治疗干预的靶点。通过这项研究，从PI3K/mTOR抑制剂文库中选择了mTORC1/2抑制剂sapanisertib与MEK抑制剂合作；后者的临床前疗效先前已被证明用于犬粘膜黑色素瘤。曲美替尼和沙巴尼替尼联合抑制MEK和mTORC1/2可导致细胞凋亡和细胞周期改变，协同降低具有不同基础信号激活水平的犬粘膜黑色素瘤细胞系的细胞存活率。与单个抑制剂相比，交错剂量的sapanisertib联合每日曲美替尼，对于限制小鼠原发性粘膜黑色素瘤异种移植物生长和转移模型中的肿瘤播散，同时最小化血液学和肾脏副作用是最佳的。抑制剂下调各自的信号靶点，并且联合抑制通路互串。两药联合的药代动力学的进一步研究正在进行中。

Research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize disease relevance produced in the model, addressing critical barriers to research progress. Additional aims include the development of new research technologies and means to interrogate cancer tissues for the evaluation and application of disease biomarkers. The research is also aimed to translate approaches to cancer treatment intended for clinical application. Progress was made in developing research resources useful in developing and characterizing new models of human cancer and in cancer diagnostics. The research resulted in methods and computational capabilities utilized to detect and quantify a metastasis promoting protein in the nuclear membrane that contributed to mechanistic insight in cell sensing and metastasis. Work in modeling breast cancer focused on elevated CLIC4 expression in human breast cancers and its alignment with early invasion and poor outcome in women. CLIC4 in the host microenvironment is required for lung metastasis, while the absence of host CLIC4, results in a microenvironment in the primary tumor and the pre-metastatic lungs that is unfavorable for tumor viability and lung colonization in the model. In particular the primary tumor microenvironment within mice deficient in CLIC4 develops necrosis and vascular abnormalities, detected through computational molecular tissue analyses. Research also focused on a highly topical and outstanding issue in tumor immunology. In pulmonary adenocarcinoma, we collaboratively posed whether the driver oncogene influences the kinds of immune cells that infiltrate the lung or their activation status. this frame of reference was undertaken for its potential clinical relevance. We profiled several lung adenocarcinomas for their immune cell infiltrations, evaluating primarily macrophages and T cell subsets, in addition to reactive tumor-associated fibroplasia in these tumors. In a small, highly curated set of samples, all specimens had some degree of immune response infiltration amid cancer cells. We did not observe a clear difference among the tumors using a limited set of parameters (CD8, CD68, CD4, FOXP3, PDL1), where mutant EGFR was the driving oncogene, versus mutant KRAS. However, the degree of fibrosis did appear to correlate with lymphocyte infiltration. This set of analyses provides a strong demonstration of the immune cell infiltration complexity in the context of tumor heterogeneity. Finally, the investigation provided correlates between presence of T regulatory lymphyocytes, high CCL5 expression and CD8+ effector function in human pulmonary adenocarcinomas. Work also continued on drug development for combined targeted therapy for mucosal melanomas. Clinical and pathological correlates between human and canine mucosal melanomas are substantial, and the relatively greater incidence of spontaneous naturally occurring mucosal melanoma in dogs represents a promising opportunity for predictive modeling. Both canine and human mucosal melanomas appear to harbor BRAF, NRAS and c-kit mutations uncommonly, compared to human cutaneous melanomas, although both species share AKT and MAPK signaling activation. The genomic landscapes of human and canine mucosal melanoma appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/AKT/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Through this research sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine mucosal melanoma. Combined inhibition of MEK and mTORC1/2, using trametinib and sapanisertib, produced apoptosis and cell-cycle alteration, synergistically reducing cell survival in canine mucosal melanoma cell lines with varying basal signaling activation levels. Compared with individual inhibitors, a staggered sapanisertib dose, coupled with daily trametinib, was optimal for limiting primary mucosal melanoma xenograft growth in mice, and tumor dissemination in a metastasis model, while minimizing hematologic and renal side effects. Inhibitors downmodulated respective signaling targets and the combination additionally suppressed pathway reciprocal crosstalk. Further studies in the pharmacokinetics of the two-drug combination are ongoing.