measuring angiogenesis in hematologic diseases maher albitar, md quest diagnostics, nichols...
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Measuring Angiogenesis in Hematologic Diseases
Maher Albitar, MD
Quest Diagnostics, Nichols Institute
San Juan Capistrano, CA 92690
(949)728-4784
Steve Potts, Ph.D.
Ravi Dayana
Allen Olson, Ph.D.
Aprio
Angiogenesis in Bone marrow
Variation in Angiogenesis Between Patients with Leukemias
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CLL2 3 874 5A
ML
6N N
VEGF
ACTIN
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Expression of Vascular Endothelial Growth Factor (VEGF) in Leukemic Cells
Angiogenesis Vs. Hematpoiesis in Hematologic Diseases:
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, .....
Antiangiogenic Agents:Antibodies: Anti-VEGF, CDP860, Cotara, Erbitux.. Small Molecule Tyrosine Kinase inhibitor: AZD2171, BAy-43-9006, PTK787/ZK, SU6668...Other: Thalidomide,…
Endothelial Cells
Autocrine
Paracrine
ParacrineLeukemia/Lymphoma Cells
Stromal Cells
-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
Groups No of No.of blood P-value Relative area P-value Corr. with patients vessels (Median) (Median %) cellularity
__________________________________________________________________________
Control 16 11.2 2.8
CLL 23 9 .56 2.4 0.78 0.45
CML 24 21.4 .003 6.2 0.02 0.33
ALL 20 18 .005 4.4 0.07 0.62
AML 30 16.1 .02 4.3 0.23 0.33
MDS 32 20.4 .004 5.2 0.06 0.74
Increased Angiogenesis in Bone Marrow in Patients with Hematologic Diseases (Manual Measurement)
High Levels of Plasma Angiogenic Factors in Leukemias and MDS
Groups VEGF bFGF
No. of
patients
pg/mL median
(range)
P-value No. of
patients
pg/mL median (range) P-value
Control 11 26.74 (23.17-53.88) 11 5.37 (4.56-13.65)
CLL 157 47.17 (22.23-720.89) <.001 155 47.77 (5.02-751.27) <.001
CML 59 76.30 (23.90-1634.8) <.001 53 8.54 (4.22-478.1) .001
ALL 28 28.16 (20.64-178.31) .21 28 7.28 (5.13-25.04) .001
AML 115 30.43 (21.47-439.25) .006 113 6.48 (4.66-203.74) .006
MDS 40 31.05 (22.45-408.7) .02 40 6.61 (4.7-49.74) .02
CMML 13 65.1 (24.33-1092) .002 19 6.54 (5.47-60.95) .001
Outcome Independent predictors of unfavorable outcome
P-value
CR VEGF 0.048 Increasing age 0.049 Zubrod performance status 3-4 0.042 Treatment with Fludarabine 0.002 DFS VEGF 0.010 -5/-7 0.003 Increasing age 0.017 Treatment with Fludarabine 0.011 Treatment in laminar air flow
room 0.027
DFS from CR
VEGF 0.002
-5/-7 <0.0001 Survival VEGF 0.019 Increasing age <0.0001 Treatment with Fludarabine 0.04 Treatment in laminar air flow
room 0.002
Intracellular VEGF is an Independent Prognostic Factor in AML and MDS
Poor ReproducibilityTime consuming and laboriousRequires pathology backgroundNot cost-effectiveOf marginal clinical ValueNo standardization
How about Automation?
Accuracy? Reproducibility?Clinically useful?Standardization between laboratories
Measuring Angiogenesis (Microvessel Density)
Define sampling area: 5-10 rectangles representative of the
sample (Automation in progress)
Automation in Measuring Angiogenesis
Calculate the space in these rectangles
Measuring Vascular Areas by Measuring Factor VIII+ Area
Highlight brown (DAB)-stainedPotentially: Define micro vessel boundary
Find lumen regions (Holes) within micro vessels
Subtracting Megakaryocytes from Measured Vascular Area
Hematoxyline stain with heavy Anti-Factor VIII are filtered out with colocalization algorithm
Find Micro Vessel BoundaryFind Lumen regionsWatershed segmentation
Double Threshold
Find & Fill Lumen Holes
Find Micro Vessels (watershed)
Measuring Partial Vascular Areas
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.
High Concordance Between Hmatopoietic Cell Number and Hematopoietic Area
R2 = 0.93
0
2
4
6
8
10
12
0 1000 2000 3000 4000
Hematopeotic cell count
Are
ar
R=0.77, P<0.0001
0.0 0.1 0.2 0.3 0.4 0.5
Automated Vascular area (%)
0
20000
40000
60000
80000
1E5
1.2E5
Ma
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asc
ula
r A
rea
Significant Correlation Between Manual and Automated Vascular Area Measurements
Vascular Area/Hematopoietic Vascular Area (%)Valid Spearman p-level Spearman p-level
Hematopoietic area 93 -0.79 0.000000 -0.42 0.000024Hematopoitic cells 93 -0.73 0.000000 -0.38 0.000181Hematopoitic/Vascular area 93 -0.95 0.000000 -0.81 0.000000Vascular area (No Meg)/Hematopoietic 93 0.79 0.000000Vascular Area (including Megs) 93 0.70 0.000000 0.96 0.000000Vascular Area (No Megs)) 93 0.79 0.000000Hematopoietic cells/area 93 -0.81 0.000000 -0.41 0.000045% Blasts 47 -0.10 0.503670 -0.18 0.227509VEGF 58 -0.04 0.760063 0.11 0.408683bFGF 39 -0.26 0.106674 -0.24 0.146458HGF 58 -0.14 0.295660 -0.01 0.967644Angiogenin 58 -0.03 0.841045 0.14 0.295660TNFa 58 0.31 0.017806 0.42 0.001167IL-6 17 0.43 0.085801 0.67 0.003310IL-8 19 0.18 0.469865 0.35 0.140402Marrow cellularity 85 -0.59 0.000000 -0.30 0.004607# of Blood Vessels (Manual) 93 0.23 0.024559 0.51 0.000000Vascular Area (manual) 93 0.61 0.000000 0.77 0.000000Relative area (Vascular/# Blood Vessels) 93 0.61 0.000000 0.77 0.000000
Correlations of Automated Angiogenesis Measurements
Clinical Correlations in Patients with Acute Myeloid Leukemia and Myelodysplastic syndrome (MDS)
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 & PLT 25 0.38 0.06 17 0.05 0.84
Vasc/Hem & % Lymph in BM 25 0.17 0.43 17 0.43 0.08
Vasc/Hem & TDT in Blasts 21 -0.35 0.12 13 0.43 0.14
Vasc/Hem & CD64 21 -0.50 0.02 12 -0.11 0.74
Vascular area (%) & TPO 10 -0.58 0.08 11 0.13 0.71
Vascular area (%) & AGE Grouping at 70 25 -0.24 0.25 17 -0.40 0.11
Vascular area (%) & Perform status 25 0.17 0.42 17 0.41 0.10
Vascular area (%) & PS grouping 25 -0.25 0.22 17 0.44 0.08
Vascular area (%) & PLT 25 0.39 0.06 17 0.00 1.00
Vascular area (%) & HGB 25 0.26 0.21 17 -0.38 0.13
Vascular area (%) & CD64 21 -0.50 0.02 12 0.06 0.85
1 3 5 7 9 11 13 15 17 19 21 23 25
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Distribution of Vascularity and Vasc/Hem in Patients with AML
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Vasc/Hem Vascularity (%)
Distribution of % Vascularity and Vasc/Hem in Patients with MDS
P=0.05
MDS (8/8; 9/9)
0 16 32 48 64 80 96 112 128 144 160 176 192
Weeks
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Cu
mu
lativ
e P
rop
ort
ion
Su
rviv
ing
<4.6% >=4.6%
Higher Vasularity (% Vascular Area) is Associated with Longer Survival in MDS
P=0.04
MDS (9/9; 8/8)
0 16 32 48 64 80 96 112 128 144 160 176 192
Weeks
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Cu
mu
lativ
e P
rop
ort
ion
Su
rviv
ing <0.23 (median Vasc/Hem)
>=0.23
Higher Vasular/Hematopoietic Area is Associated with Longer Survival in MDS
0 52 104 156 208 260 312 364
Weeks
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
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ing
No correlation between Angiogenesis and Survival in AML
CRD, P=0.04
CRD and Angigenesis % in AML (5/3; 6/6)
0 52 104 156 208 260 312
Weeks
0.0
0.2
0.4
0.6
0.8
1.0
Cu
mu
lativ
e P
rop
ort
ion
Su
rviv
ing
<0.48 (median) >= 0.48
Higher Vasular/Hematopoietic Area is Associated with Shorter Remission Duration
in AML
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 cellularityVascular 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