1. Measuring Angiogenesis in Hematologic Diseases
Maher Albitar, MD
Quest Diagnostics, Nichols Institute
San Juan Capistrano, CA 92690
(949)
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 N N 1 2 3 4 5 6 7 8 kD 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 , PTK787/ZK, SU
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. Clinical Correlations in Patients with Acute Myeloid Leukemia and Myelodysplastic syndrome (MDS)
AML
MDS
Valid
R-value
p-level
Vasc/Hem & Days to respond 25
0.24
0.26 17
0.50
0.04
Vasc/Hem & Cytogenetic groups
0.35
0.08
-0.22
0.40
Vasc/Hem
& PLT
0.38
0.06
0.05
0.84
& % Lymph in BM
0.17
0.43
Vasc/Hem &
TDT in Blasts 21
-0.35
0.12 13
0.14
& CD64
-0.50
0.02 12
-0.11
0.74
Vascular area (%)
& TPO 10
-0.58 11
0.13
0.71
& AGE Grouping at 70
-0.24
0.25
-0.40
0.11
& Perform status
0.42
0.41
0.10
& PS grouping
-0.25
0.22
0.44
0.39
0.00
1.00
& HGB
0.21
-0.38
0.85

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