Zhengyu Pang

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

The relative distribution of membranous and cytoplasmic met is a prognostic indicator in stage I and II colon cancer.

Purpose: The association hepatocyte growth factor receptor (Met) tyrosine kinase with prognosis and survival in colon cancer is unclear, due in part to the limitation of detection methods used. In particular, conventional chromagenic immunohistochemistry (IHC) has several limitations including the inability to separate compartmental measurements. Measurement of membrane, cytoplasm, and nuclear levels of Met could offer a superior approach to traditional IHC. Experimental Design: Fluorescent-based IHC for Met was done in 583 colon cancer patients in a tissue microarray format. Using curvature and intensity-based image analysis, the membrane, nuclear, and cytoplasm were segmented. Probability distributions of Met within each compartment were determined, and an automated scoring algorithm was generated. An optimal score cutpoint was calculated using 500-fold crossvalidation of a training and test data set. For comparison with conventional IHC, a second array from the same tissue microarray block was 3,3′-diaminobenzidine immunostained for Met. Results: In crossvalidated and univariate Cox analysis, the membrane relative to cytoplasm Met score was a significant predictor of survival in stage I (hazard ratio, 0.16; P = 0.006) and in stage II patients (hazard ratio, 0.34; P ≤ 0.0005). Similar results were found with multivariate analysis. Met in the membrane alone was not a significant predictor of outcome in all patients or within stage. In the 3,3′-diaminobenzidine–stained array, no associations were found with Met expression and survival. Conclusions: These data indicate that the relative subcellular distribution of Met, as measured by novel automated image analysis, may be a valuable biomarker for estimating colon cancer prognosis.

A novel, automated technology for multiplex biomarker imaging and application to breast cancer

Multiplexed immunofluorescence is a powerful tool for validating multigene assays and understanding the complex interplay of proteins implicated in breast cancer within a morphological context. We describe a novel technology for imaging an extended panel of biomarkers on a single, formalin‐fixed paraffin‐embedded breast sample and evaluating biomarker interaction at a single‐cell level, and demonstrate proof‐of‐concept on a small set of breast tumours, including those which co‐express hormone receptors with Her2/neu and Ki‐67.

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes.

Autofluorescence removal using a customized filter set

Quantitative fluorescence microscopy is severely hindered by intrinsic autofluorescence (AF). Endogenous fluorescent molecules in tissue and cell samples emit fluorescence that often dominates signals from specific dyes. This makes AF removal critical to the development and practice of quantitative fluorescence microscopy. In this study, we showed that AF signal could be separated from specific signal using a customized filter set. The filter set used the same excitation and beam splitter as the standard filter set, but the emission filter was red‐shifted 40–60 nm from the peak of the specific dye. This filter set configuration collected mostly AF with minimum contribution from the specific dye. A linear transformation of AF images was required to correct for the difference in exposure and filter configuration. The constants (slope and intercept) in linear transformation were obtained through a pixel to pixel comparison between AF images (no staining) obtained by the standard filter set and the customized AF filter set. After staining of specific dye, the standard filter collecting target dye spectra was used to capture both target signal and AF, whereas customized filter was used to capture only AF. AF removal was accomplished by subtracting the linear transformed AF image from the image obtained from the standard filter. To validate our approach, we examined weak staining of androgen receptor in an AF abundant prostate tissue sample. Our method revealed a similar but cleaner nuclear staining of androgen receptor in a specimen, when compared to a traditional autofluorescence removal method. Microsc. Res. Tech., 76:1007–1015, 2013.

Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.

In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, Na+K+-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed.

Epithelial Cell Segmentation via Shape Ranking

We present a robust and high-throughput computational method for cell segmentation using multiplexed immunohistopathology images. The major challenges in obtaining an accurate cell segmentation from tissue samples are due to (i) complex cell and tissue morphology, (ii) different sources of variability including non-homogeneous staining and microscope specific noise, and (iii) tissue quality. Here we present a fast method that uses cell shape and scale information via unsupervised machine learning to enhance and improve general purpose segmentation methods. The proposed method is well suited for tissue cytology because it captures the the morphological and shape heterogeneity in different cell populations. We discuss our segmentation framework for analysing approximately one hundred images of lung and colon cancer and we restrict our analysis to epithelial cells.

Abstract 2499: Application of a multiplexed fluorescence microscopy method (MultiOmyxTM) to dissect proteomic biomarkers of (18)F-fluorodeoxy-glucose ((18)FDG) uptake in breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: While routinely collected human tumor specimen can now readily be examined for genetic alterations on a genome wide scale, multi-parameter measurements of protein expression remain challenging. We developed a multiplexed fluorescence microscopy method (MultiOmyxTM) for the quantitative characterization of multiple analytes in formalin-fixed paraffin-embedded tissue (PMID23818604). We now applied this platform to human breast cancer samples to determine the expression of 25 proteins at a single-cell level and examine their relationship to tumor uptake of the Positron Emission Tomography (PET) radiotracer (18)F-fluorodeoxy-glucose ((18)FDG). Methodology: We stained a single 5 µm section of breast carcinoma from each of 18 patients with antibodies against members of growth factor signaling pathways (HER2, IGF1R, PTEN, p-EGFR, p-PDK1, p-ERK1/2, p-S6 Ribosomal Protein, p-4EBP1, p-eIF4E), the glycolysis pathway (Glut-1, HK2, LDH-A), hormone receptors (ER, PR, AR), tumor cell proliferation markers (KI-67 and phospho-histone H3), and markers of hypoxia (HIF-1α, CA IX), and angiogenesis (CD31). 28-30 representative fields of view (FOVs) were randomly selected in each tumor, each comprising 300-500 cells. Images were automatically separated into subcellular and histopathological compartments based on the staining of tumor cells with a panel of segmentation markers (NaK-ATPase, pan-Cadherin, pan-cytokeratin, S6, and DAPI). A breast pathologist assessed and annotated the histologic composition of each FOV. Further analysis focused on FOVs (n=390) containing only invasive ductal carcinoma without admixed DCIS or normal breast tissue. Multivariate analysis between marker expression and FDG uptake was performed using logistic regression and Cox proportional hazard models. All patients had (18)-FDG PET within four weeks prior to collection of the tumor specimen. Results: Staining results with our MultiOmyxTM platform correlated closely with the results from CLIA-certified single marker biomarker assays (e.g., ER IHC and HER2 FISH assay) performed independently on the same set of samples (PMID: 21646475). Nuclear ER staining was associated with low FDG uptake (p=0.02). KI-67 was higher in tumors with high FDG uptake (p=0.04). K-median clustering identified molecular breast cancer subtypes. Conclusions: Our study illustrates the feasibility of quantitative proteomic measurements using a new in-situ multiplexed fluorescence microscopy platform on only a single routinely collected FFPE tissue section. Quantitative staining results from many thousand cells per tumor support the previously reported relationship between hormone receptor status, tumor cell proliferation, and FDG-uptake in breast cancer. Multiple protein markers showed a high degree of intra- and inter-tumoral heterogeneity. Citation Format: Anup Sood, Alexandra M. Miller, Fiona Ginty, Elizabeth McDonough, Yunxia Sui, Alexander Bordwell, Qing Li, Sireesha Kaanumalle, Zhengyu Pang, Franklin Torres, Edi Brogi, Steven Larson, Ingo Mellinghoff. Application of a multiplexed fluorescence microscopy method (MultiOmyxTM) to dissect proteomic biomarkers of (18)F-fluorodeoxy-glucose ((18)FDG) uptake in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2014-2499

Abstract A28: Multiplexed immunofluorescence in formalin-fixed paraffin-embedded prostate cancer

Traditional immunohistochemistry serves as a vital diagnostic assay as it allows for semiquantitatively probing the magnitude and spatial distribution of protein expression and posttranslational modifications. Unfortunately, an accurate portrayal of disease often requires several immunohistochemical stains, which are run on separate tissue sections. The application of transcriptomics analyses in cancer classification has led to the generation of multivariate index analyses that confer diagnostic, prognostic, and predictive signatures of disease. However this method is limited by the inability consider target expression in the context of tissue (stroma, epithelium) or cell (e.g. stem/progenitor cells) type. A unifying platform that would allow for the quantitative spatial analysis of multiplexed in situ target measurements promises to exploit the best features of both systems. To this end, we have developed a novel multiplexed fluorescence immunohistochemistry assay that is capable of quantitative analysis of >25 antigens in a single formalin-fixed paraffin-embedded tissue section, as well as image and data analysis capabilities for analyzing the multidimensional data. By inclusion of targets for cell and tissue compartments, including nuclei, cytoplasm and membrane, we have demonstrated the ability to investigate subcellular protein expression, phosphorylation and co-localization in individual cells. In the present study we have stained a cohort of 79 prostate cancer patients of varying Gleason grades for a total of 24 antigens, representing key mediators of cell signaling, tumor suppressors and oncogenes, tumor associated phenotypic targets, and structural proteins. The images are precisely registered and segmented into individual cells nuclear, cytoplasmic and membrane regions and the underlying expression of a given antigen is then quantitatively determined in each compartment by automated algorithms. With the advent of this new tool, we aim to define new prognostic molecular signatures in prostate cancer. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A28.