Lieser, Elizabeth Ann T.

Activated platelets release mitogenic factors, vascular remodeling enzymes, inflammatory and cellular adhesive molecules into the vascular microenvironment to alter chemotactic and adhesive properties of vascular endothelium. Platelet‐derived growth factor‐BB (PDGF‐BB) and matrix metalloproteinase‐2 (MMP‐2) increased in the platelets following ovariectomy but decreased with estrogen treatment in pigs. Experiments were designed to identify changes in these factors using custom antibody arrays in lysate of platelets collected from women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic. Washed platelets were prepared from anticoagulated blood before (baseline) and 48 months after randomization into either placebo or oral or transdermal MHT. Preliminary analysis shows after 48 months compared to baseline, platelet content of C‐reactive protein, intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), PDGF‐BB, MMP‐2 and transforming growth factor‐beta (TGF‐beta) were lower (20–60%) in two groups but higher (20–74%) in one group. Based on our studies in pigs, it is expected that the two groups showing decreases in platelet proteins would be from women randomized to MHT. However, KEEPS will not be unblinded until February 2012. (Supported by grants from Aurora Foundation, NIH HL90639, 1UL1 RR024150).

8599 Background: Chemotherapy resistance is a common, difficult problem for melanoma patients needing long term care and those suffering from recurrence. Neural growth factor receptor (NGFR), also known as CD271, is commonly expressed on mesenchymal stem cells and is important for development, survival and differentiation of cells. This has previously shown to be expressed on melanoma stem cells. We hypothesized that treatment with carboplatin enriches the population of tumor cells for cancer stem cells (CSC) as a mechanism of chemotherapy resistance. Methods: Core tumor samples from 118 patients with metastatic melanoma were obtained from formalin-fixed paraffin embedded specimens. Slides were stained using an anti-CD271 antibody for IHC. A375 melanoma cells were treated with increasing concentrations of carboplatin for approximately 1 year. WT, 6 mg/mL and 10 mg/mL resistant cells were tagged with CD271-PE antibody and run on a flow cytometer. These cells were then tested for gene expression by microarray using an Affymetrix human 133 plus 2.0 chip, and analyzed on Partek software. Pathway analysis of these carboplatin resistant cells was preformed using Ingenuity. Results: Virtually all melanoma tumor samples stained positively for CD271 by IHC, with a median of 50% cells within tumor samples expressing the cytoplasmic marker. Flow cytometry demonstrated an increase in the percentage of cells expressing CD271 as resistance to carboplatin increased. Wild type A375 cells contained 30% positive CD271, A375 6mg/mL resistant contained 42% positive and A375 10mg/mL resistant showed 66% positive. Microarray also exhibited a direct correlation between CD271 gene expression with increasing carboplatin resistance. Functional ontology enrichment revealed development and regulation of the EMT pathway as an important process impacted by our resistant cell line. Conclusions: The data suggests that as melanoma cells become resistant to certain chemotherapy drugs they essentially loose their differentiated phenotype and become more stem cell-like. Monitoring melanoma expression of CD271 could help individualize therapy and anticipate chemotherapeutic resistance in this high-risk group of patients.