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Preliminary Validation of a Multispectral Image Analysis Application for Confirmation
of Isolated Tumor Cells in Axillary Lymph Nodes from Breast Cancer Patients
Jeffrey Fine MD, K McManus, A Luketich, D Dabbs MD
University of Pittsburgh
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Objectives
• Background– Hard to Stain Breast Cancer Metastases– Multispectral Image Analysis
• Clinical Application & Validation
• Path Forward
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Sentinel Lymph Node Biopsy(Breast Cancer)
• Surgeon identifies axillary nodes most likely to contain metastatic disease
• If negative, no further biopsy needed– Low probability of unsampled disease
• If positive…it depends on how positive
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Multispectral Image Analysis (MSI)
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Multispectral Image Analysis (MSI)
• Image based on spectral information instead of color
• Each pixel has a spectrum instead of a color
• This data permits “demixing” the ‘colors’
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Light Sensitivity
Wavelength
RGB
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Light Sensitivity
Wavelength
7 Bands
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How to Stain Very Small Foci
• Try a traditional stain (it might work)
• Destain the H&E and Immunostain that slide
• Immunostain the H&E directly then use MSI to demix the colors and create both H&E and Immunostain images
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Application
• Perform Cytokeratin Immunostain onto H&E– “multiplex” H&E and IHC
• Use MSI to produce FALSE COLOR IMAGES– Pseudo H&E– Pseudo IHC
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Details
• Macrometastases for initial validation– Only 5 cases to start
• Immunostaining– AE1/AE3 antibody (Dako)– Benchmark XT (Ventana)
• MSI– Nuance System (CRi)
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Suspense Spared
• Validated relative to traditional stains– Positive and negative controls
• Stain not as brilliant but visible
• Stain process bleached H&E—re-staining required (H, E, and DAB staining required to create good false color images)
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Workflow
• Pathologist orders an MSI protocol, gives slide to IHC lab
• AE 1/3 stain performed on H&E (details omitted)
• Slide given to Pathologist (for now) for MSI
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Results
• False color H&E and AE1/3 images returned to pathologist
• …Slide returned as well
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Why…
…is the slide returned?– Easily verifiable by eye—no need for blind trust
…lymph nodes?– Uncommon (not rare) frustrating situation
…bother?
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Why do MSI in these cases?
• Introduction of MSI technology into semi-routine surgical pathology workflow
• Demonstration of technology to other pathologists
• Development of MSI workflow for other applications
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Challenges
• Original H&E still “destroyed”– Whole Slide Image archival
• Limited field of view—cannot MSI the entire slide (foci must be marked as with FISH slides)
• Demixing is very far from perfect– IHC pretty good but digital H&E is in progress
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Presentation
• Demixing limitations– Crosstalk prevents creation of higher quality false
color H&E
• Presentation– Optimal false color combinations need tweaking
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Next Steps
• Longer semi-validation phase– Continue attempting regular IHC on these cases– Return original slide to pathologist for validation
• Publicize availability to increase volume– First me; then select others; then everybody
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Next Applications
• Breast—microinvasion (myoepithelial markers)
• Prostate biopsies – small foci
• Greater “automation” – performance by technical staff
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Concluding Thoughts
• Available NOW (MSI today)– Imperfect but it validates– Leads directly to other similar applications
• This can drive improvements– General image analysis workflow (inc delegation)– Better algorithms– Experience (currently more of an art)
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