1. PATIENT CHARECTERISTICS Pair-Wise Comparisons
A Predictive Biomarker of Vasculopathy in Survivors of Childhood Malignancies
Pradhan K.. MBBS, MS; Claussen H. CCRP; Vik T.. MD; Calley C.. MS; Mund J.. MS; Case J.. PhD
Indiana University School of Medicine and Riley Hospital for Children, Indianapolis, Indiana
BACKGROUND
CONCLUSIONS
METHODS
VASCULAR-REACTIVITY TESTING
► Cardiovascular and Cerebrovascular late-effects occur in survivors of childhood cancer at increased frequency compared to general population.
► Cranial radiotherapy, anthracyclines, mediastinal and heart radiation are the main offending agents.
► Survivors of Hodgkin's lymphoma, acute leukemia, and brain-tumors are at highest risks for these vascular accidents.
► Underlying mechanisms of these injuries are understudied but thought to be due to endothelial injury mitigated by pro-inflammatory cytokines and reactive oxygen species.
► Although multiple screening modalities for early detection of these late-effects have been recommended, there are no established peripheral blood (PB) biomarkers that correlate with vascular dysfunction.
► Our exploratory study was designed to detect the frequency of circulating cellular markers of angiogenesis in cancer-survivors with the goal to eventually establish them as biomarkers of vascular dysfunction.
NOVEL CIRCULATING CELLS WITH ANGIOGENIC POTENTIAL
► These are circulating hematopoietic stem and progenitor cells (CHSPCs), also called circulating progenitor cells (CPCs).
► CPCs are hematopoietic in origin based on their expression of CD45, CD34 and CD31.
► CPCs are further characterized into two sub-populations based on expression of AC133. The pro-angiogenic CPCs (pCPCs) are CD31+CD34brightCD45dimAC133+, and the non-angiogenic CPCs (nCPCs) are CD31+CD34brightCD45dimAC133-.
► A polychromatic flow cytometry (PFC) protocol was developed and used to isolate these novel CPCs and their sub-sets1.
► In patients with peripheral arterial disease (PAD) the ratio of pCPCs to nCPCs is reduced to 0.8±0.16% (mean ±SEM) compared to age and sex-matched controls (1.81±0.09% mean ±SEM)1.
► We used PFC to measure CPCs and their subsets in children with cancer and found the ratio of pCPCs to nCPCs elevated in cancer-patients compared to healthy controls2.
► Based on the above mentioned data we hypothesized that the ratio of the pCPCs to nCPCs would be lower in cancer-survivors who had on going endothelial dysfunction but were clinically well, indicating it might help in detection of “silent patients” harboring vascular dysfunction.
► PB was collected in CPT tubes and mononuclear cells (MNCs) were isolated and stained with CD34, AC133, CD45, CD31, CD14, CD41a, CD235a as well as a viability marker for the exclusion of dead cells.
►A dump channel was used to exclude non-viable cells, red blood cells and platelets from analysis.
►The gating strategies are shown below. Fluorescence minus one (FMO) controls were prepared as gating controls to allow proper gating of true positive populations.
►Stained MNC samples were fixed and run on a BD LSR II flow-cytometer equipped with a 405nm, 488m, and 633nm laser configuration. Data was analyzed using FlowJo software version (TreeStar).
►The results for each individual cell-type were expressed as percentage of total circulating MNCs1.
► Participant’s forearms were measured using a probe connected to a laser doppler instrument as published in previous reports.
► Basal skin perfusion was measured and then either acetylcholine or sodium nitroprusside was introduced into the skin using intophoresis to measure the endothelium independent and endothelium dependent vasodilatation respectively.
► Percent change in perfusion was calculated every 20 seconds at 60-second intervals3.
► This vascular-reactivity is utilized as a surrogate marker of endothelial health.
► CPCs and the pCPC subset are likely candidates to stimulate and maintain physiological angiogenesis through paracrine mechanisms with interactions with other pro-angiogenic cells and proteins including the vascular endothelium to maintain normal vascular homeostasis.
► This is the first study to demonstrate low levels of CPCs and the pCPC to nCPC ratio among survivors of childhood-cancers. Circulating endothelial cells (CECs) were also low in cancer-survivors compared to healthy controls. CECs are live mature endothelial cells found in the peripheral blood.
► This implies that some cancer-survivors potentially at risk for vascular dysfunction exhibit low levels of CPCs and the decreased pCPC to nCPC ratio.
► We compared the vascular-reactivity of the vascular endothelium to acetylcholine and sodium nitroprusside between the survivors and healthy controls and found no difference between the two groups.
► Therefore the total number of CPC and pCPC to nCPC ratio were already affected even before endothelial dysfunction and might aid in early detection of survivors who are in an asymptomatic phase of vascular-dysfunction.
PATIENT CHARECTERISTICS
Diagnosis
Sex
Age (yrs)
Time since Rx
(yrs)
XRT
SCT
ALL M 27 22
Yes
---
Lymphoma-NHL 28 11 No
auto
Medullobastoma 16
CNS-PNET 23 7 19 F 15 6
CNS-Germ Cell 8
allo
Wilms 12 20 13
Neuroblastoma
Hodgkins 29
FUTURE-DIRECTIONS
A prospective, longitudinal, cross-sectional study of cancer-survivors treated with radiation therapy v/s anthracyclines is being planned.
A genotype-phenotype co-relation between “target-genes” and cellular markers of angiogenesis may shed more light on “personalizing” cancer treatment in the future by reducing therapies in those at highest risk for vascular dysfunctions.
REFERENCES
RESULTS
Application of Polychromatic Flow Cytometery to Identify Novel Subsets of Circulating Cells with Angiogenic Potential. Estes et al., Cytometry Part A. 77A: , 2010.
Polychromatic Flow Cytometry Identifies Novel Subsets of Circulating Cells With Angiogenic Potential in Pediatric Solid Tumors. Pradhan et al., Cytometry Part B. 80B: , 2011.
Impaired Skin Microvascular Function in Children, Adolescents, and Young Adults with Type 1 Diabetes. Khan et al., Diabetes Care 23: , 2000.
Novel Cellular
Markers
Overall Comparison
Pair-Wise Comparisons
Kruskal-Wallis
p-value
Cancer Patients
vs. Healthy Controls
Cancer Survivors
vs. Controls
vs. Cancer Patients
CPC
<0.0001
0.2175
0.0003
0.0006
ECFC
0.5863 --
CEC
0.0111
0.1808
0.0057
0.2917
pCPC/nCPC
0.0033
0.1328
0.0009
0.0478
ACKNOWLEDGEMENTS
This study was supported by the Division of Radiation-Oncology, IU Simon Cancer Center and the Division of Pediatric Hematology Oncology, Riley Hospital for Children, Indiana University Health, Indianapolis, IN.
We acknowledge the Angiogenesis, Endothelial and Pro-Angiogenic Cell Core (AEPCC) of the Indiana University Simon Cancer Center for the processing and PFC analysis of the PB samples for this study.