Steven J. Potts

Evaluating tumor heterogeneity in immunohistochemistry-stained breast cancer tissue

Quantitative clinical measurement of heterogeneity in immunohistochemistry staining would be useful in evaluating patient therapeutic response and in identifying underlying issues in histopathology laboratory quality control. A heterogeneity scoring approach (HetMap) was designed to visualize a individual patients immunohistochemistry heterogeneity in the context of a patient population. HER2 semiquantitative analysis was combined with ecology diversity statistics to evaluate cell-level heterogeneity (consistency of protein expression within neighboring cells in a tumor nest) and tumor-level heterogeneity (differences of protein expression across a tumor as represented by a tissue section). This approach was evaluated on HER2 immunohistochemistry-stained breast cancer samples using 200 specimens across two different laboratories with three pathologists per laboratory, each outlining regions of tumor for scoring by automatic cell-based image analysis. HetMap was evaluated using three different scoring schemes: HER2 scoring according to American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) guidelines, H-score, and a new continuous HER2 score (HER2cont). Two definitions of heterogeneity, cell-level and tumor-level, provided useful independent measures of heterogeneity. Cases where pathologists had disagreement over reads in the area of clinical importance (+1 and +2) had statistically significantly higher levels of tumor-level heterogeneity. Cell-level heterogeneity, reported either as an average or the maximum area of heterogeneity across a slide, had low levels of dependency on the pathologist choice of region, while tumor-level heterogeneity measurements had more dependence on the pathologist choice of regions. HetMap is a measure of heterogeneity, by which pathologists, oncologists, and drug development organizations can view cell-level and tumor-level heterogeneity for a patient for a given marker in the context of an entire patient cohort. Heterogeneity analysis can be used to identify tumors with differing degrees of heterogeneity, or to highlight slides that should be rechecked for QC issues. Tumor heterogeneity plays a significant role in disconcordant reads between pathologists.

The role and impact of quantitative discovery pathology

The decision to advance an early-stage compound into formal preclinical testing depends on confidence in mechanism, efficacy and toxicity profiles. A substantial percentage of this confidence comes from histopathology interpretation, as the local tissue environment contains strong signals of both efficacy and toxicity. Accessing this tissue information is made difficult by biological variability across organs and tissues, an insufficient pool of pathology experts working in discovery, and the high subjectivity and individual isolation of microscope-based observations. This article describes how whole-slide imaging and quantitative analysis by trained pathologists are improving early-stage decision-making.

Digital pathology in drug discovery and development: multisite integration

Digital pathology is an emerging technology that provides an image-based environment for managing and interpreting the information generated from a digitized glass slide, offering substantial improvements in pharmaceutical drug development across discovery, preclinical GLP pathology and oncology clinical trials. Digital pathology is transforming global pharmaceutical research by enabling data sharing to integrate dispersed pharma pathology labs around the world. This article reviews the stages of multisite digital pathology integration in large pharmaceutical companies, offering suggestions for success and highlighting challenges.

Multiplexed measurement of proteins in tissue in a clinical environment.

There is an emerging need for more effective approaches to accurately quantitate protein expression in tissue samples. In many clinical studies and particularly in pharmaceutical clinical trials, access to adequate tissue samples is a major bottleneck, and thus techniques to measure protein expression in these valuable tissue specimens is important. This study will review current approaches in multiplexing of protein expression in tissue, and discusses new approaches using a novel image registration technique across multiple tissue sections.

Tissue pattern recognition error rates and tumor heterogeneity in gastric cancer.

The anatomic pathology discipline is slowly moving toward a digital workflow, where pathologists will evaluate whole-slide images on a computer monitor rather than glass slides through a microscope. One of the driving factors in this workflow is computer-assisted scoring, which depends on appropriate selection of regions of interest. With advances in tissue pattern recognition techniques, a more precise region of the tissue can be evaluated, no longer bound by the pathologist’s patience in manually outlining target tissue areas. Pathologists use entire tissues from which to determine a score in a region of interest when making manual immunohistochemistry assessments. Tissue pattern recognition theoretically offers this same advantage; however, error rates exist in any tissue pattern recognition program, and these error rates contribute to errors in the overall score. To provide a real-world example of tissue pattern recognition, 11 HER2-stained upper gastrointestinal malignancies with high heterogeneity were evaluated. HER2 scoring of gastric cancer was chosen due to its increasing importance in gastrointestinal disease. A method is introduced for quantifying the error rates of tissue pattern recognition. The trade-off between fully sampling tumor with a given tissue pattern recognition error rate versus randomly sampling a limited number of fields of view with higher target accuracy was modeled with a Monte-Carlo simulation. Under most scenarios, stereological methods of sampling-limited fields of view outperformed whole-slide tissue pattern recognition approaches for accurate immunohistochemistry analysis. The importance of educating pathologists in the use of statistical sampling is discussed, along with the emerging role of hybrid whole-tissue imaging and stereological approaches.

Molecular histopathology and tissue biomarkers in drug and diagnostic development

The aims of diagnostic and therapeutic development are to accurately diagnose and cure disease, respectively. For the past century and a half, histopathology—the microscopic examination of cells and tissues—has been considered a “gold standard” for the diagnosis of many diseases. As an introduction to this volume on molecular histopathology and tissue biomarkers in drug and diagnostic development, I explore the relationship between histopathology and the nature of disease itself. A lack of agreement on the meaning of “disease” has led to widespread and indiscriminate use of the term. Here, I propose that the term “disease” be reserved for conditions where there exists some knowledge of alterations in cells or their products that participate in cause-effect relationships in lesional (diseased) tissue. This is a definition that simultaneously lends legitimacy to the term’s use while enabling revision and testing of hypotheses based on rapidly emerging scientific knowledge. With this perspective, histopathology, as a preferred means to visualize and depict the cellular events that constitute disease as it impacts tissue structure and function, will remain essential to develop new diagnostic tests and targeted therapies for the foreseeable future.

AngioMap is a novel image analysis algorithm for assessment of plasma cell distribution within bone marrow vascular niche.

The ability to characterize distribution of neoplastic hematopoietic cells and their progenitors in their native microenvironment is emerging as an important challenge and potential therapeutic target in many disease areas, including multiple myeloma. In multiple myeloma, bone marrow (BM) angiogenesis is typically increased and microvessel density is a known indicator of poor prognosis. However, the difficulty of consistently measuring 3D vessels from 2D cut sections has previously limited the study of spatial distribution of plasma cells (PC) and their interaction with BM microenvironment. The aim of the study is to report a novel method to study myeloma cells spatial distribution within their hematopoietic niche context using readily available tissue sections and standard histology approaches. We utilized a novel whole-tissue image analysis approach to identify vessels, and then applied computational grown regions extended out from each vessel at 15, 35, 55, 75, and 100 &mgr;m to identify the spatial distribution of PC on CD34/CD138 double-stained core biopsy slides. Percent PC to total cells (TC) was significantly higher at <15 &mgr;m distance compared with those at 16 to 35, 36 to 55, 56 to 75, and 76 to 100 &mgr;m distance (P=0.0001). Similarly, PC/TC at <35 &mgr;m region was significantly higher compared with 36 to 55 (P=0.0001), 56 to 75 (P⩽0.0001), and 76 to 100 (P=0.0002) &mgr;m distances. The mean PC/TC differences in the spatial gradient of 36 to 55, 56 to 75, and 76 to 100 &mgr;m distance regions were not significant. Our findings suggest possible preferential advantage to neoplastic PC in the proximity of blood vessels compared with other hematopoietic marrow cells. We demonstrate the feasibility of analyzing the spatial distribution of PC, and possibly other hematopoietic/stem cells in their microenvironment, as characterized by the distance to vessels in BM using a novel image analysis approach.

Measuring Protein Expression in Tissue The Complementary Roles of Brightfield and Fluorescence in Whole Slide Scanning

Reproducible protein expression measurement is important in many areas of tissue research and must address the inherently high biological variability across a single tissue section. Whole slide imaging is a new technology offering substantial improvements for both brightfield and fluorescence measurement of protein expression. In this article we discuss the latest developments in fluorescence and brightfield whole slide imaging as it applies to protein expression, and discuss how the 2 approaches differ and are complementary.

Validation of an image analysis algorithm to quantify leukocyte populations using whole slide image analysis

In recent years, the tumor microenvironment (TME) has been identified as an important factor influencing the growth and metastasis of the tumor. In the TME, different classes of inflammatory cells have been found to exert either a pro- or anti-tumor effect. This has resulted in a growing need to utilize immunohistochemistry to label these leukocyte populations, thereby allowing for the cells to be quantified. In many instances these studies have been performed utilizing 2-3 independent readers to manually quantify the cells requiring significant time both for the actual counting as well as the training needed to minimize the variation from reader to reader. In addition, manual counting is usually done on selective high-powered fields rather than the entire specimen, resulting in variations in counts when different fields are chosen. A key method to increase the throughput and to decrease the variability is to utilize whole slide imaging and computerized image analysis to provide leukocyte counts. An image analysis algorithm which can automatically differentiate tumor from stroma would allow rapid quantification of endpoints in each compartment, such as: tumor burden; number of inflammatory cells/area; or percent of inflammatory cells/total cells in each tissue compartment. In this poster, the validation and utilization of an algorithm to quantify immunolabeled leukocytes in both tumor sections and tissue microarrays is described. Utilizing whole slide imaging approaches, an image analysis algorithm (CellMap™) that allows the quantitation of leukocyte populations (e.g., CD3+, CD8+, FoxP3+) automatically across whole tissue sections has been developed. This approach has been used to evaluate samples of colorectal cancer and non-small cell lung carcinoma. Using this algorithm, leukocyte populations were quantified in sections that have been either singly or dually labeled for inflammatory markers. Accuracy of the algorithm was demonstrated by comparing data from manual counts to algorithm derived counts using high-powered fields. The results of the high-powered field analysis were compared to an analysis across the whole tissue section, demonstrating the effect of variability when user defined fields are chosen. These data support using CellMap™ in the prospective or retrospective assessment of leukocyte subpopulations in clinical samples. This approach will diminish variability in counting, expand the types of endpoints determined, and improve the statistical value of these determinations, thereby facilitating robust TME measurements with clinical value.

Abstract 5543: Hetmap: Evaluating tumor heterogeneity in immunohistochemistry-stained breast cancer tissue

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Immunohistochemisty assays examining the HER2 receptor in breast cancer is the most widely adopted example of a companion diagnostic approach, which seeks to dictate therapeutic strategy based on a molecular description of a patients disease. There are well-established guidelines for selecting patients for anti HER2 adjuvant therapies in breast cancer treatment, yet the current HER2 companion diagnostic approach is qualitative, does not sufficiently account for intratumor heterogeneity, and does not utilize any additional information about tumor cells that score beyond a specific threshold level. A major contributing factor for the failure of both treatment and diagnostic paradigm is thought to be intratumor heterogeneity. This lack of information in the scoring paradigm may contribute to inappropriate patient selection and explain why the disease in many trastuzumab treated patients progresses or becomes recurrent. Thus, a quantitative clinical measurement of heterogeneity in immunohistochemistry staining would be useful in better predicting patient therapeutic response. To answer this, we created a heterogeneity scoring approach (HetMap) that allows the visualization of an individual patients IHC heterogeneity in the context of a cell population. We combined HER2 semi-quantitative analysis with the use of ecology diversity statistics to evaluate cell-level heterogeneity (consistency of protein expression within neighboring cells in a tumor nest) and tumor-level heterogeneity (differences of protein expression across a tumor as represented by a tissue section). We evaluated the approach on HER2 immunohistochemistry stained breast cancer samples, using 200 specimens across two different CLIA laboratories, with three pathologists at each laboratory each outlining regions of tumor for scoring by automatic cell-based image analysis. HetMap was evaluated using three different scoring schemes: HER2 scoring according to ASCO/CAP guidelines, H-Score and a new continuous HER2 score (HER2cont). Cell-level heterogeneity, reported either as an average or the maximum area of heterogeneity across a slide, had low levels of dependency on the pathologist choice of region. Tumor-level heterogeneity measurements had more dependence on the pathologist choice of regions. Significantly, discordant pathologist assessments of IHC scores in the +2, or equivocal score, range occurred most in tumors with high heterogeneity, leading to potentially major clinical impact. Thus, HetMap is a measure of heterogeneity, by which pathologists, oncologists, and drug development organizations can view cell-level and tumor-level heterogeneity for a patient for a given marker in the context of an entire patient cohort. Including such measures of heterogeneity in diagnostic approaches can help establish better thresholds for patient selection, thereby improving patient response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5543. doi:1538-7445.AM2012-5543