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

Objectives

• Background– Hard to Stain Breast Cancer Metastases– Multispectral Image Analysis

• Clinical Application & Validation

• Path Forward

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

Multispectral Image Analysis (MSI)

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’

Light Sensitivity

Wavelength

RGB

Light Sensitivity

Wavelength

7 Bands

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

Application

• Perform Cytokeratin Immunostain onto H&E– “multiplex” H&E and IHC

• Use MSI to produce FALSE COLOR IMAGES– Pseudo H&E– Pseudo IHC

Details

• Macrometastases for initial validation– Only 5 cases to start

• Immunostaining– AE1/AE3 antibody (Dako)– Benchmark XT (Ventana)

• MSI– Nuance System (CRi)

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)

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

Results

• False color H&E and AE1/3 images returned to pathologist

• …Slide returned as well

Why…

…is the slide returned?– Easily verifiable by eye—no need for blind trust

…lymph nodes?– Uncommon (not rare) frustrating situation

…bother?

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

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

Presentation

• Demixing limitations– Crosstalk prevents creation of higher quality false

color H&E

• Presentation– Optimal false color combinations need tweaking

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

Next Applications

• Breast—microinvasion (myoepithelial markers)

• Prostate biopsies – small foci

• Greater “automation” – performance by technical staff

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)