Abstract 1454: Evaluation of negative selection strategy for enrichment of circulating melanoma cells (CMCs) from patients with metastatic melanoma.
Authors: Joshi Powrnima , Jacobs Barbara , Derakhshan Adeeb , Moore Lee R. , Triozzi Pierre , Borden Ernest C. , Zborowski Maciej 
Published: August 2014

Abstract
Introduction. Circulating tumor cells (CTCs) have emerged as a predictive biomarker in the treatment of metastatic breast, colorectal and prostate cancers. Non-epithelial tumors such as melanoma also present evidence of circulating cells that have been difficult to detect due to technological limitations. Previous clinical studies have shown presence of melanoma specific marker mRNAs circulating in the blood, used as a surrogate of the circulating melanoma cell (CMC). This study was designed to use a negative cell selection platform to enrich CMCs from blood. The approach involved depletion of red blood cells by centrifugation and lysis, and white blood cells by immunomagnetic tagging with pan-leukocyte (CD45) antibody and magnetic separation. The enriched non-hematopoietic cell analysis was optimized for high sensitivity and specificity of CMC detection using a panel of antibodies against known melanoma markers. The secondary goal was to estimate the number of CMCs per milliliter of blood based on immunophenotype and morphology criteria.
Methods. The study was reviewed and approved by the IRB. The leukocyte depletion was accomplished using a fluid dynamics-based quadrupole magnetic separating device (1) which, in principle, enriches CMCs without affecting them by immunomagnetic reagents. CMC cytospin smears were immunostained against known melanoma markers (Melan-A and S100). A high magnification microscopy imaging and a computer-aided image analysis were developed for efficient CMC enumeration.
Results. Based on preliminary studies with SKMEL-28 melanoma cell line, normal adult blood controls and blood samples from advanced melanoma patients, the CMC was defined as an intact, nucleated cell, positive for Melan A or S100 and negative for CD45. Separations of SKMEL-28 cells spiked at approximately one SKMEL-28 cell per million leukocytes resulted in high efficiency separation with a mean recovery of (93.0 ±5.4 S.E.M.)% of SKMEL-28 cells. Samples obtained from apparently healthy donor blood (n =5) had a baseline of 0-3 positive cells per mL blood, whereas samples from stage IV melanoma patients (n =8) exhibit CMC counts ranging from 78 to 28,500 cells per mL of blood.
Conclusions. Our study combines negative selection platform with immunophenotypic and morphological criteria for CMC enrichment, identification and enumeration. Interestingly, our preliminary results indicate the presence of high numbers of CMCs in malignant melanoma patients, providing basis for larger studies on correlations with clinical outcomes of the disease. Moreover, negative selection method lends itself to in vitro or in vivo culture bioassays, and molecular marker assays such as qRT-PCR, genomic PCRs, FISH or microarrays.
Citation Format: Powrnima Joshi, Barbara Jacobs, Adeeb Derakhshan, Lee R. Moore, Pierre Triozzi, Ernest C. Borden, Maciej Zborowski. Evaluation of negative selection strategy for enrichment of circulating melanoma cells (CMCs) from patients with metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1454. doi:10.1158/1538-7445.AM2013-1454