Measuring Angiogenesis in Hematologic Diseases

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Measuring Angiogenesis in Hematologic Diseases
Maher Albitar, MD Quest Diagnostics, Nichols
Institute San Juan Capistrano, CA
92690 [phone] Maher.x.Albitar_at_Questdiagnost
ics.com Steve Potts, Ph.D. Ravi Dayana Allen
Olson, Ph.D. Aprio
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Angiogenesis in Bone marrow
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Variation in Angiogenesis Between Patients with
Leukemias
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Expression of Vascular Endothelial Growth Factor
(VEGF) in Leukemic Cells
CML-Plasma
CML-Plasma
CML-Cells
CML-Cells
CLL
AML
kD
8
7
5
1
2
3
4
6
N
N
57
45
VEGF
43
38
ACTIN
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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
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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
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High Levels of Plasma Angiogenic Factors in
Leukemias and MDS
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Intracellular VEGF is an Independent Prognostic
Factor in AML and MDS
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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

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Automation in Measuring Angiogenesis

• Define sampling area
• 5-10 rectangles representative of the sample
  (Automation in progress)

Calculate the space in these rectangles
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Measuring Vascular Areas by Measuring Factor
VIII Area
Highlight brown (DAB)-stained
Potentially Define micro vessel boundary
Find lumen regions (Holes) within micro vessels
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Subtracting Megakaryocytes from Measured Vascular
Area
Hematoxyline stain with heavy Anti-Factor VIII
are filtered out with colocalization algorithm
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Measuring Partial Vascular Areas
Find Micro Vessels (watershed)
Find Fill Lumen Holes
Double Threshold
Find Micro Vessel Boundary Find Lumen
regions Watershed segmentation
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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.
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High Concordance Between Hmatopoietic Cell Number
and Hematopoietic Area
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Significant Correlation Between Manual and
Automated Vascular Area Measurements
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Correlations of Automated Angiogenesis
Measurements
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Clinical Correlations in Patients with Acute
Myeloid Leukemia and Myelodysplastic syndrome
(MDS)
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Distribution of Vascularity and Vasc/Hem in
Patients with AML
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Distribution of Vascularity and Vasc/Hem in
Patients with MDS
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Higher Vasularity ( Vascular Area) is Associated
with Longer Survival in MDS
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Higher Vasular/Hematopoietic Area is Associated
with Longer Survival in MDS
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No correlation between Angiogenesis and Survival
in AML
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Higher Vasular/Hematopoietic Area is Associated
with Shorter Remission Duration in AML
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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