1. 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

2. Objectives Background – Hard to Stain Breast Cancer Metastases – Multispectral Image Analysis Clinical Application & Validation Path Forward

3. 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

10. Multispectral Image Analysis (MSI)

15. Image based on spectral information instead of color Each pixel has a spectrum instead of a color This data permits “demixing” the ‘colors’

16. Light Sensitivity Wavelength RGB

17. Light Sensitivity Wavelength 7 Bands

18. 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

25. Application Perform Cytokeratin Immunostain onto H&E – “multiplex” H&E and IHC Use MSI to produce FALSE COLOR IMAGES – Pseudo H&E – Pseudo IHC

26. Details Macrometastases for initial validation – Only 5 cases to start Immunostaining – AE1/AE3 antibody (Dako) – Benchmark XT (Ventana) MSI – Nuance System (CRi)

28. 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)

43. 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

44. Results False color H&E and AE1/3 images returned to pathologist …Slide returned as well

45. Why… …is the slide returned? – Easily verifiable by eye—no need for blind trust …lymph nodes? – Uncommon (not rare) frustrating situation …bother?

46. 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

47. 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

48. Presentation Demixing limitations – Crosstalk prevents creation of higher quality false color H&E Presentation – Optimal false color combinations need tweaking

56. 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

57. Next Applications Breast—microinvasion (myoepithelial markers) Prostate biopsies – small foci Greater “automation” – performance by technical staff

58. 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)