The current standard in pathology is to measure biomarkers singly by IHC and in situ hybridization with visual inspection and manual scoring or at best simple digital imaging of single biomarkers. Digital pathology slide scanners and IHC image analysis applications are increasing in adoption in medical laboratories; however, there is a need for faster slide scanning and for multiplexed approaches to quantify multiple key biomarkers in the same tissue section to improve pathology testing. The use of an H and E tissue section image is important as an aid to pathologists, since this is the standard of practice and the value of the pathologists′ interpretation of the tissue morphology, especially in conjunction with multiple biomarkers, is essential. Fluorescence digital imaging pathology with image analysis algorithms will enable objective, quantitative analysis of multiple biomarkers per slide to meet this need to incorporate novel multivariate biomarker tests into pathology testing. We investigated the potential applications of combined brightfield and fluorescence digital slide imaging using the NanoZoomer Digital Pathology slide scanner with HCImage image analysis software. This system has various potential clinical applications and advantages versus traditional pathology methods and over other digital pathology systems. The slide scanner digitizes slides at exceptional speed in both brightfield and fluorescence imaging modes, which make it an ideal slide scanning solution for both research and clinical pathology applications. There are various brightfield digital slide scanning systems available from multiple vendors; however, scanning speed is a significant barrier to clinical implementation due to the need for short turnaround times in pathology testing. The NanoZoomer used in this study is faster than any other fluorescence slide scanner on the market and Hamamatsu is further improving the scanning speed in a second-generation version of the NanoZoomer (NanoZoomer 2.0-HT (C9600-13)). Fluorescence slide scanning systems are also being introduced to meet the need for multiplexed biomarker analysis in pathology. Fluorescence slide scanners that image 4-6 fluorescence channels individually are available based on multipass single sensor approaches. The slower speed of these scanners and the lack of FDA-approved image analysis algorithms for specific clinical applications make them currently suitable only for research. Adoption of fluorescence digital imaging pathology will continue to increase as improvements are made to the scanning speed and as clinical applications are developed and validated.
The NanoZoomer Digital Pathology system combines fluorescence digital imaging of multiple biomarkers on one slide with brightfield digital imaging of tissue morphology and includes image analysis software (HCImage) to objectively extract quantitative data on multiple biomarkers per tissue section. The dual brightfield – fluorescence capabilities will be a useful aid to pathologists to assess fluorescence biomarker images in the context of the tissue morphology. In this study we demonstrated quantitative analysis of multiplexed biomarkers in example tissues and control cells lines in TMA format. The HCImage image analysis software-enabled segmentation of individual cells within tissues into subcellular compartments such that we were able to measure the mean and total intensity of biomarkers in nuclei and the cytoplasm and plasma membrane of single cells within tissues and measure the ratio of biomarker intensity between the different subcellular compartments. These measurements have various potential clinical applications since for many biomarkers the expression, tissue localization, subcellular localization, and ratio between different tissue and cellular compartments are relevant to their diagnostic, prognostic and/or predictive significance[19
] (reviewed in[8
]). The software includes TMA segmentation and extracted tissue core organization to accommodate high-throughput analysis of multiple biomarkers in hundreds to thousands of patient samples per slide for biomarker assay development and validation studies.
The applications of the NanoZoomer Digital Pathology system include combined transmitted light and 3-color multiplexed fluorescence quantitative biomarker assays in research and clinical pathology. Multiple separate IHC biomarker scores have been used to stratify cancer patients according to prognosis;[23
] however, there are difficulties scoring and interpreting multiple IHC stains in single cells within tissues and this tedious manual method of measuring many markers singly with manual scoring is neither realistic in terms of efficiency nor reproducible clinically. Digital pathology has been applied to IHC analysis to improve objectivity and reproducibility.[25
] Even with digital analysis, the accuracy of IHC is only semiquantitative and is limited by inherent variability in staining intensity and the difficulties in multiplexing. Multiplexed fluorescence with digital imaging offers significant improvements on IHC, including: (i) more consistent, higher resolution in the labeling and imaging since fluorophores are directly conjugated to antibodies, (ii) greater dynamic detection range, (iii) the ability to quantify multiple antigens per section, including multiple antigens colocalized within the same subcellular compartment, (iv) objective, reproducible extraction of quantitative biomarker data by image analysis software. Quantitative fluorescence biomarker analysis has been shown to correlate with Western blot analysis,[27
] to match or exceed the accuracy of manual IHC scoring,[28
] and to improve standardization of tissue biomarker testing.[31
The NanoZoomer system has a 3-color/multiplexed biomarker limit, due to the detection method. However, there are many current and potential future clinical applications that require only 2-3 biomarkers coupled to a reference H&E image test, e.g., cytokeratins and/or Ki-67 in various tumor types, estrogen receptor (ER), progesterone receptor (PgR) and HER2-/neu in breast cancer or p63, high molecular weight cytokeratin, and P504S in prostate cancer. Using three simultaneous sensors for fluorescence scanning as the NanoZoomer does is very efficient for such applications. Novel multivariate index biomarker tests are likely to be based on 6--10, or possible even higher numbers of biomarkers. Higher multiplexing can be achieved by staining and imaging biomarkers in serial sections using the NanoZoomer. The ideal system for tissue-based multivariate index assays would accommodate all the relevant biomarkers on one slide. There is a challenge for next-generation systems to provide the optimal trade-off between scanning speed and number of fluorescence channels. In order to overcome the single exposure time for each channel with the NanoZoomer, it is critical to optimize the immunofluorescence biomarker staining to improve signal:noise in each fluorescence channel.
In summary, the NanoZoomer Digital Pathology system with HCImage image analysis software enables high-throughput brightfield imaging of tissue morphology and fluorescence imaging of multiple biomarkers per slide with quantitative, objective image analysis at the subcellular compartments. The system has potential applications in 3-color fluorescence biomarker testing to improve the objectivity and efficiency of pathology testing and in high-throughput analysis of biomarkers in research pathology.