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Measuring Angiogenesis in Hematologic Diseases

Measuring Angiogenesis in Hematologic Diseases. Maher Albitar, MD Quest Diagnostics, Nichols Institute San Juan Capistrano, CA 92690 (949)728-4784 Maher.x.Albitar@Questdiagnostics.com Steve Potts, Ph.D. Ravi Dayana Allen Olson, Ph.D. Aprio. Angiogenesis in Bone marrow.

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Measuring Angiogenesis in Hematologic Diseases

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  1. Measuring Angiogenesis in Hematologic Diseases Maher Albitar, MD Quest Diagnostics, Nichols Institute San Juan Capistrano, CA 92690 (949)728-4784 Maher.x.Albitar@Questdiagnostics.com Steve Potts, Ph.D. Ravi Dayana Allen Olson, Ph.D. Aprio

  2. Angiogenesis in Bone marrow

  3. Variation in Angiogenesis Between Patients with Leukemias

  4. Expression of Vascular Endothelial Growth Factor (VEGF) in Leukemic Cells CML-Plasma CML-Plasma CML-Cells CML-Cells CLL AML kD 5 7 8 N N 1 2 3 4 6 57 45 VEGF 43 38 ACTIN

  5. Angiogenesis Vs. Hematpoiesis in Hematologic Diseases: Antiangiogenic Agents: Antibodies: Anti-VEGF, CDP860, Cotara, Erbitux.. Small Molecule Tyrosine Kinase inhibitor: AZD2171, BAy-43-9006, PTK787/ZK, SU6668... Other: Thalidomide,… Proangiogenic/Anti-angiogenic Factors: VEGF (multiple forms), bFGF, tal-1, Cox2, tie1, PIGF, PD-ECGF, HGF, MMP, PDGF, TNF-a, Angiogenin, TGF-a/b, G-CSF, VEGFR1/VEGFR2, NP1/NP2, ..... Autocrine Paracrine Endothelial Cells Paracrine Leukemia/Lymphoma Cells Stromal Cells

  6. Increased Angiogenesis in Bone Marrow in Patients with Hematologic Diseases (Manual Measurement) • -IHC: FVIII antigen-positive endothelial cells • -Digitized images are analyzed for: • 1. Number of blood vessels/10 fields (200X) • 2. Lumen area/10 fields (200X) using NIH Image software

  7. High Levels of Plasma Angiogenic Factors in Leukemias and MDS

  8. Intracellular VEGF is an Independent Prognostic Factor in AML and MDS

  9. Measuring Angiogenesis (Microvessel Density) • Poor Reproducibility • Time consuming and laborious • Requires pathology background • Not cost-effective • Of marginal clinical Value • No standardization How about Automation? • Accuracy? • Reproducibility? • Clinically useful? • Standardization between laboratories

  10. Automation in Measuring Angiogenesis • Define sampling area: • 5-10 rectangles representative of the sample (Automation in progress) Calculate the space in these rectangles

  11. Measuring Vascular Areas by Measuring Factor VIII+ Area Highlight brown (DAB)-stained Potentially: Define micro vessel boundary Find lumen regions (Holes) within micro vessels

  12. Subtracting Megakaryocytes from Measured Vascular Area Hematoxyline stain with heavy Anti-Factor VIII are filtered out with colocalization algorithm

  13. Measuring Partial Vascular Areas Find Micro Vessels (watershed) Find & Fill Lumen Holes Double Threshold Find Micro Vessel Boundary Find Lumen regions Watershed segmentation

  14. Measuring Vascular Area relative to Hematopoietic Area/Cells (Vasc/Hem) Hematoxyline-stained area or individual cells can be counted in the same rectangles tested for angiogenesis.

  15. High Concordance Between Hmatopoietic Cell Number and Hematopoietic Area

  16. Significant Correlation Between Manual and Automated Vascular Area Measurements

  17. Correlations of Automated Angiogenesis Measurements

  18. AML MDS Valid R-value p-level Valid R-value p-level Vasc/Hem & Days to respond 25 0.24 0.26 17 0.50 0.04 Vasc/Hem & Cytogenetic groups 25 0.35 0.08 17 -0.22 0.40 Vasc/Hem 25 0.38 0.06 17 0.05 0.84 & PLT Vasc/Hem 25 0.17 0.43 17 0.43 0.08 & % Lymph in BM Vasc/Hem & 21 -0.35 0.12 13 0.43 0.14 TDT in Blasts Vasc/Hem 21 -0.50 0.02 12 -0.11 0.74 & CD64 Vascular area (%) & TPO 10 -0.58 0.08 11 0.13 0.71 Vascular area (%) 25 -0.24 0.25 17 -0.40 0.11 & AGE Grouping at 70 Vascular area (%) & Perform status 25 0.17 0.42 17 0.41 0.10 Vascular area (%) 25 -0.25 0.22 17 0.44 0.08 & PS grouping Vascular area (%) 25 0.39 0.06 17 0.00 1.00 & PLT Vascular area (%) 25 0.26 0.21 17 -0.38 0.13 & HGB Vascular area (%) 21 -0.50 0.02 12 0.06 0.85 & CD64 Clinical Correlations in Patients with Acute Myeloid Leukemia and Myelodysplastic syndrome (MDS)

  19. Distribution of Vascularity and Vasc/Hem in Patients with AML

  20. Distribution of % Vascularity and Vasc/Hem in Patients with MDS

  21. Higher Vasularity (% Vascular Area) is Associated with Longer Survival in MDS

  22. Higher Vasular/Hematopoietic Area is Associated with Longer Survival in MDS

  23. No correlation between Angiogenesis and Survival in AML

  24. Higher Vasular/Hematopoietic Area is Associated with Shorter Remission Duration in AML

  25. Summary • Measuring Vascularity may Provide a surrogate marker for clinical behavior • Automation in measuring angiogenesis is a reliable means for standardizing measurement of angiogenesis • Automation allows the measurement of: Vascular Area (%) Hematopoietic cellularity Vascular Area relative to cellularity (Vasc/Hem) • Larger studies are needed for clinical and biological correlations • Further studies are needed to correlate with response the anti-angiogenic therapy

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