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T Definiens Tissue StudioTM 2.0 Definiens Oncology Suite

The Most Advanced Digital Pathology Image Analysis Solution For Biomarker Translational Research – Just Got Better.

New in Definiens Tissue Studio™ 2.0

Full immunofluorescence support Improved nucleus detection Customized “tuning” of Definiens Composer™ technology Faster performance And still – simple to use!

Definiens Tissue Studio™ 2.0 – the solution of choice for tumor biomarker and morphological profiling Definiens Tissue Studio™ 2.0 is the solution of choice for tumor biomarker and morphological profiling because it is easy to use, accurate, robust, powerful and fast.

Definiens Tissue Studio™ 2.0 provides localized, cell-by-cell biomarker analysis; as well as relational and multiparametric morphological quantification of cells and sub-cellular structures. Whether you are working with bright field or immunofluorescence images; on whole tissue slides or tissue microarrays; Definiens Tissue Studio™ 2.0 is your ultimate resource for comprehensive digital pathology image analysis.

Figure 1 Definiens Tissue Studio™ 2.0 now fully supports immunofluorescent image quanti­fication. In this example, nuclei are detected in xenograft tissue stained for DAPI, CD206, CD68 and a therapeutic antibody candidate. Up to 12 channels are supported.

Understanding Images

Definiens Tissue Studio™ 2.0 supports most commercial image formats Definiens Tissue Studio™ 2.0 supports native image file formats from most commercially available digital slide image acquisition systems. This enables laboratories to standardize on one image analysis platform improving productivity and reproducibility. Definiens Tissue Studio™ 2.0 also supports .tiff, .jpeg, .bmp and .jpeg2000.

Figure 2 Definiens Tissue Studio™ 2.0 dialogue window for selecting image file formats from most commercially available image acquisition platforms.

A single platform for bright field and immunofluorescence image analysis Simply start the program, and choose whether you want to analyze bright field or immunofluorescence images; in either slide or tissue microarray formats. There is no need for additional software.

Figure 3 After launching the program, the user selects four analysis modes: Bright field (slides or TMAs); or immunofluorescence (slides or TMAs)

Definiens Tissue Studio™ 2.0 provides a simple to use, 4-step workflow Load, Configure, Run, Review. It is that easy. New users can expect to develop a customized image analysis solution in about 20 minutes. Simply load your training

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images, configure your solution, run the solution across your image set, and review the results. The exported results are contained within easy to access folders and include per-object information, classified images and more.

Figure 4 The simple 4-step workflow in Definiens Tissue Studio™ 2.0 – Load, Configure, Run, and Review.

The Analysis Builder is the heart of Definiens Tissue Studio™ 2.0. It combines advanced image analysis technology with a simple and efficient user interface; providing flexibility for configuring robust solutions over a wide variety of stains and tissue types. The user sets parameters for detecting regions of interest; cellular and sub-cellular analysis within these regions; and various data export options.

Figure 5 The Analysis Builder’s simple color coded configuration panel makes it easy to develop a customized image analysis solution.

Region of interest detection with Definiens Composer™ technology – Interactive, simple, accurate, and reliable Definiens Composer™ technology is an easy to use method for generating robust region of interest detection. In this “learn by example” format, you simply use the paintbrush tool to “brush” on representative regions of interest, and then click the “learn” button. It is that easy. New in Definiens Tissue Studio™ 2.0 is the ability to tune Definiens Composer™ technology for even more precise region of interest detection.

Figure 6 The Definiens Composer™ technology control panel. New in Definiens Tissue Studio™ 2.0 is the “select features” button which allows for fine tuning the performance of Definiens Composer™ technology.

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Figure 7 Definiens Composer™ technology in action – In this image, all staining for Her2 in breast tumor tissue has been classified in orange; surrounding tissue, yellow; and white space, blue.

For manual annotation, the Magnetic Snap tool helps to follow the contours of a region of interest; making manual annotation quick and easy. The Magnetic Snap tool can be used in combination with Definiens Composer™ technology.

Figure 8 The Magnetic Snap tool in action

Definiens Tissue Studio™ 2.0 allows accurate detection of cells and sub-cellular components After region of interest training with Definiens Composer™ technology, subsets are selected (i.e., from tumor regions), to configure the system to properly identify cells and sub-cellular objects (membrane, cytoplasm, nuclei). It only takes a few interactive steps to properly adjust the software for accurate detection of cells and sub-cellular structures.

Figure 9a Definiens Tissue Studio™ 2.0 preview of the detection of cells and sub-cellular structures in breast tis­sue. In this step, the user interactively adjusts the software until the desired result is achieved.

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The next configuration step is to set the staining thresholds for biomarker quantification. Although the software makes suggestions for default low, medium, and high thresholds, this step is ultimately user defined which ensures accurate cell-by-cell evaluation of biomarker expression.

Figure 9b Definiens Tissue Studio™ 2.0 staining intensity preview (low, medium, or high). As in figure 9a, the user interactively adjusts the software until the desired result is achieved. The solution is saved and batch processing can now begin.

Definiens Tissue Studio™ 2.0 is also able to exclude cells from the analysis. In Figures 9a and 9b, all cells where nuclei were not present have been excluded from further analysis. You can exclude cells based on area, length, width, distance from border, etc.

The most comprehensive biomarker and morphological quantification available for digital pathology image analysis Increasingly, the trend in the literature shows that in addition to localized biomarker expression, tumor morphology plays a role in disease prognosis or therapeutic response prediction. Definiens Tissue Studio™ 2.0 provides the world’s most comprehensive digital pathology image analysis for tumor morphological quantification.

Figure 10 The image object table and image object information tables are displayed. Clicking on any object (cell, nucleus, cytoplasm, region of interest, etc.) will yield all quantified features for that particular object.

Length, area, width, elliptic fit, circularity, and dozens more intensity-, shape- and context-based features can be measured. You can even create your own attributes to quantify, and there is no limit to how many parameters can be measured or exported. Further, there is no “speed limit” for comprehensive image analysis processing. Turnaround times are the same whether you are measuring marker intensity; percent stain; percent positive or negative nuclei; or for example, 15 different morphological attributes of an individual cell.

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Figure 11 Example data generated from a typical Definiens Tissue Studio analysis. Over 50 intensity-, shape-, and context-based features are available.

Full tissue microarray support for retrospective studies Tissue microarrays are often utilized for retrospective studies involving tumor biomarker and morphological profiling. In these settings, researchers look for correlations between biomarker expression, tumor morphology, and clinical endpoints of interest. Definiens Tissue Studio™ 2.0 provides full tissue microarray support, for both bright field and immunofluorescent images, and supports metadata import on a per core basis. Even with heavily skewed tissue microarrays, Definiens Tissue Studio™ 2.0 easily matches cores.

Figure 12 a-d In these images, a non-small cancer tissue microarray was analyzed for the nuclear expression of SOX2. The study concluded that SOX2 over-expression correlates to longer survival in patients with squamous non-small cell lung cancer (Images courtesy of Sven Perner and Theresia Wilbertz; University of Tuebingen).

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Definiens Tissue Studio™ 2.0 delivers:

DEEPER INSIGHTS Quantifies nuclear, membrane and cytoplasmic biomarkers on a cell-by-cell basis

Supports all standard bright field and immunofluorescent stains

Analyzes all slides and TMA images from most commercially available image acquisition systems

FASTER RESULTS Identifies regions of interest automatically

Operates through a simple, intuitive user interface

Offers unlimited throughput with parallel batch processing

BETTER DECISIONS Delivers extremely accurate and reproducible results

Enables multiparametric investigations to identify underlying correlations

Supports translational research through rapid iterative investigation of biomarkers

Multiparametric and morphological features measured IHC and IF

No. of positive nuclei

No. of negative nuclei

Positive index

Cell and membrane features: – Mean cell size (μm²) – Mean marker intensity (Cell) – Mean marker intensity (Cytoplasm) – Mean marker intensity (Membrane) – Mean ratio marker intensity

membrane to cytoplasm Marker area features: – IHC: Marker area (μm²) – Fluorescence: For up to three chosen channels as well as co-localization areas:

– Marker area (μm²) – Marker intensity

No. and % of: – Nuclei of low intensity – Nuclei of medium intensity – Nuclei of high intensity

No. and % of: – Nuclei with small area – Nuclei with medium area – Nuclei with large area No. and % of: – Cells of negative intensity (0) – Cells of low intensity (1+) – Cells of medium intensity (2+) – Cells of high intensity (3+)

No. and % of: – Cells with small area – Cells with medium area – Cells with large area

H & E Region of interest area

Custom data export is also supported: Over 50 intensity, shape, and context related features are available including: Area, length, width, length/ width, roundness, compactness, density, circularity, elliptic fit, shape index, and many more.

See http://www.tissuestudio.com for the complete feature listing.

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System requirements

Definiens Tissue Studio™ 2.0 is available as a workstation for either two or four analysis engines to meet your processing requirements. Additionally, it is available as part of the Definiens XD enterprise solution, which allows the installation of separate client and server components. The system requirements for the workstation configurations are below:

DEFINIENS TISSUE STUDIO™ 2.0 TWO ANALYSIS ENGINE CONFIGURATION Minimum: Recommended:

• Windows XP-64Bit, Vista-64 Bit, Windows 7-64 Bit • Windows XP-64 Bit, Vista-64 Bit, Windows 7-64 Bit

• Intel / AMD Quad Core 64 Bit 1.83GHz • Intel Xeon/ AMD Quad Core 64 Bit 2.00GHz

• 8 GB Memory • 12 GB Memory (1.33GHz Bus)

• 30 GB Disk Space • 30 GB Disk Space

• 20“ 1600 x 1200 Display • 24“ 1920 x 1200 Display +

• 100 MB Ethernet network connection • 1GB Ethernet Connection

DEFINIENS TISSUE STUDIO™ 2.0 FOUR ANALYSIS ENGINE CONFIGURATION

Minimum: Recommended:

• Windows XP-64Bit, Vista-64 Bit, Windows 7-64 Bit • Windows XP-64 Bit, Vista-64 Bit, Windows 7-64 Bit

• Intel / AMD Dual CPU (Socket), • Intel Xeon/ AMD Dual CPU (Socket), Quad Core 64 Bit 1.83GHz + Quad Core 64 Bit 2.00GHz+

• 12 GB Memory • 16 GB Memory (1.33GHz Bus)

• 30 GB Disk Space • 30 GB Disk Space

• 20“ 1600 x 1200 Display • 24“ 1920 x 1200 Display +

• 100 MB Ethernet network connection • 1GB Ethernet Connection

Definiens is the number one Enterprise Image Intelligence® company for analyzing and interpreting images on every scale, from microscopic cell structures to full body scans.

The patented Definiens Cognition Network Technology®, developed by Nobel Laureate Prof. Gerd Binnig and his team, emulates human cognitive processes of perception to extract intelligence from images. If you are interested in learning more about how Definiens could address the challenges you face, please visit our website.

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All registered trademarks, pending trademarks, or service marks are property of their respective owners. The information in this document is subject to change with­out notice and should not be construed as a commitment by Definiens AG. Definiens AG assumes no responsibility for any errors that may appear in this document.