Quantitative Image Analysis for Tissue Biomarker Use: A White Paper From the Digital Pathology Association.

Tissue biomarkers have been of increasing utility for scientific research, diagnosing disease, and treatment response prediction. There has been a steady shift away from qualitative assessment toward providing more quantitative scores for these biomarkers. The application of quantitative image analysis has thus become an indispensable tool for in-depth tissue biomarker interrogation in these contexts. This white paper reviews current technologies being employed for quantitative image analysis, their application and pitfalls, regulatory framework demands, and guidelines established for promoting their safe adoption in clinical practice.

Discovery of TAK-981, a First-in-Class Inhibitor of SUMO-Activating Enzyme for the Treatment of Cancer.

SUMOylation is a reversible post-translational modification that regulates protein function through covalent attachment of small ubiquitin-like modifier (SUMO) proteins. The process of SUMOylating proteins involves an enzymatic cascade, the first step of which entails the activation of a SUMO protein through an ATP-dependent process catalyzed by SUMO-activating enzyme (SAE). Here, we describe the identification of TAK-981, a mechanism-based inhibitor of SAE which forms a SUMO-TAK-981 adduct as the inhibitory species within the enzyme catalytic site. Optimization of selectivity against related enzymes as well as enhancement of mean residence time of the adduct were critical to the identification of compounds with potent cellular pathway inhibition and ultimately a prolonged pharmacodynamic effect and efficacy in preclinical tumor models, culminating in the identification of the clinical molecule TAK-981.

MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy.

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.

Localization of human TACC3 to mitotic spindles is mediated by phosphorylation on Ser558 by Aurora A: a novel pharmacodynamic method for measuring Aurora A activity.

Aurora A is a serine/threonine protein kinase essential for normal mitotic progression. Aberrant increased expression of Aurora A, which occurs frequently in human cancers, results in abnormal mitoses leading to chromosome instability and possibly tumorigenesis. Consequently, Aurora A has received considerable attention as a potential target for anticancer therapeutic intervention. Aurora A coordinates several essential mitotic activities through phosphorylation of a variety of proteins, including TACC3, which modulates microtubule stabilization of the mitotic spindle. Recent studies identified a conserved serine in Xenopus (Ser(626)) and Drosophila (Ser(863)) TACC3 orthologues that is phosphorylated by Aurora A. We show that this conserved serine on human TACC3 (Ser(558)) is also phosphorylated by Aurora A. Moreover, phosphorylation of TACC3 by Aurora A in human cells is essential for its proper localization to centrosomes and proximal mitotic spindles. Inhibition of Aurora A with the selective small molecule inhibitor MLN8054 in cultured human tumor cells resulted in mislocalization of TACC3 away from mitotic spindles in a concentration-dependent manner. Furthermore, oral administration of MLN8054 to nude mice bearing HCT-116 human tumor xenografts caused a dose-dependent mislocalization of TACC3 away from spindle poles that correlated with tumor growth inhibition. As TACC3 localization to mitotic spindles depends on Aurora A-mediated phosphorylation, quantifying TACC3 mislocalization represents a novel pharmacodynamic approach for measuring Aurora A activity in cancer patients treated with inhibitors of Aurora A kinase.

A Practical Guide to Whole Slide Imaging: A White Paper From the Digital Pathology Association.

CONTEXT.­: Whole slide imaging (WSI) represents a paradigm shift in pathology, serving as a necessary first step for a wide array of digital tools to enter the field. Its basic function is to digitize glass slides, but its impact on pathology workflows, reproducibility, dissemination of educational material, expansion of service to underprivileged areas, and intrainstitutional and interinstitutional collaboration exemplifies a significant innovative movement with far-reaching effects. Although the benefits of WSI to pathology practices, academic centers, and research institutions are many, the complexities of implementation remain an obstacle to widespread adoption. In the wake of the first regulatory clearance of WSI for primary diagnosis in the United States, some barriers to adoption have fallen. Nevertheless, implementation of WSI remains a difficult prospect for many institutions, especially those with stakeholders unfamiliar with the technologies necessary to implement a system or who cannot effectively communicate to executive leadership and sponsors the benefits of a technology that may lack clear and immediate reimbursement opportunity. OBJECTIVES.­: To present an overview of WSI technology-present and future-and to demonstrate several immediate applications of WSI that support pathology practice, medical education, research, and collaboration. DATA SOURCES.­: Peer-reviewed literature was reviewed by pathologists, scientists, and technologists who have practical knowledge of and experience with WSI. CONCLUSIONS.­: Implementation of WSI is a multifaceted and inherently multidisciplinary endeavor requiring contributions from pathologists, technologists, and executive leadership. Improved understanding of the current challenges to implementation, as well as the benefits and successes of the technology, can help prospective users identify the best path for success.

Introduction to Digital Image Analysis in Whole-slide Imaging: A White Paper from the Digital Pathology Association.

The advent of whole-slide imaging in digital pathology has brought about the advancement of computer-aided examination of tissue via digital image analysis. Digitized slides can now be easily annotated and analyzed via a variety of algorithms. This study reviews the fundamentals of tissue image analysis and aims to provide pathologists with basic information regarding the features, applications, and general workflow of these new tools. The review gives an overview of the basic categories of software solutions available, potential analysis strategies, technical considerations, and general algorithm readouts. Advantages and limitations of tissue image analysis are discussed, and emerging concepts, such as artificial intelligence and machine learning, are introduced. Finally, examples of how digital image analysis tools are currently being used in diagnostic laboratories, translational research, and drug development are discussed.

A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts.

Objective. Use of tyramide signal amplification (TSA) to detect autophagy biomarkers in formalin fixed and paraffin embedded (FFPE) xenograft tissue. Materials and Methods. Autophagy marker regulation was studied in xenograft tissues using Amp HQ IHC and standard IHC methods. Results. The data demonstrate the feasibility of using high sensitivity TSA IHC assays to measure low abundant autophagy markers in FFPE xenograft tissue.

Discovery of a potent and orally bioavailable benzolactam-derived inhibitor of Polo-like kinase 1 (MLN0905).

This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic-pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.

Phase I assessment of new mechanism-based pharmacodynamic biomarkers for MLN8054, a small-molecule inhibitor of Aurora A kinase.

The mitotic kinase Aurora A is an important therapeutic target for cancer therapy. This study evaluated new mechanism-based pharmacodynamic biomarkers in cancer patients in two phase I studies of MLN8054, a small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received MLN8054 orally for 7 consecutive days in escalating dose cohorts, with skin and tumor biopsies obtained before and after dosing. Skin biopsies were evaluated for increased mitotic cells within the basal epithelium. Tumor biopsies were assessed for accumulation of mitotic cells within proliferative tumor regions. Several patients in the highest dose cohorts showed marked increases in the skin mitotic index after dosing. Although some tumors exhibited increases in mitotic cells after dosing, others displayed decreases, a variable outcome consistent with dual mechanisms of mitotic arrest and mitotic slippage induced by antimitotics in tumors. To provide a clearer picture, mitotic cell chromosome alignment and spindle bipolarity, new biomarkers of Aurora A inhibition that act independently of mitotic arrest or slippage, were assessed in the tumor biopsies. Several patients, primarily in the highest dose cohorts, had marked decreases in the percentage of mitotic cells with aligned chromosomes and bipolar spindles after dosing. Evidence existed for an exposure-effect relationship for mitotic cells with defects in chromosome alignment and spindle bipolarity that indicated a biologically active dose range. Outcomes of pharmacodynamic assays from skin and tumor biopsies were concordant in several patients. Together, these new pharmacodynamic assays provide evidence for Aurora A inhibition by MLN8054 in patient skin and tumor tissues.

Phase I study of the selective Aurora A kinase inhibitor MLN8054 in patients with advanced solid tumors: safety, pharmacokinetics, and pharmacodynamics.

This phase I trial examined the safety, pharmacokinetics, and pharmacodynamics of MLN8054, an oral, selective, small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received increasing doses of MLN8054 in 28-day cycles until dose-limiting toxicity (DLT) was seen in ≥2 of 3-6 patients in a cohort. For the 10-mg and 20-mg cohorts, treatment was administered once daily on days 1 to 5 and 8 to 12. Patients in later cohorts (25, 35, 45, 55, 60, 70, and 80 mg/day) were treated four times daily on days 1 to 14, with the largest dose at bedtime (QID-14D) to mitigate benzodiazepine-like effects possibly associated with peak plasma concentrations. Patients (n = 43) received a median of 1 cycle (range, 1-10). DLT of somnolence was first noted in the 20-mg cohort. Two DLTs of somnolence (n = 1) and transaminitis (n = 1) were seen at QID-14D 80 mg. Grade 2 oral mucositis (n = 1), predicted to be a mechanistic effect, was observed only at QID-14D 80 mg. MLN8054 exposure levels were roughly linear with dose; terminal half-life was 30 to 40 hours. Pharmacodynamic analyses of skin and tumor mitotic indices, mitotic cell chromosome alignment, and spindle bipolarity provided evidence of Aurora A inhibition. MLN8054 dosing for 10 to 14 days in 28-day cycles was feasible. Somnolence and transaminitis were DLTs. Pharmacodynamic analyses in mitotic cells of both skin and tumor provided proof of mechanism for Aurora A kinase inhibition. A more potent, selective, second-generation Aurora A kinase inhibitor, MLN8237, is in clinical development.