Isolation and expansion of circulating tumor cells (CTC) from melanoma patients using a novel cell culture technique.

Abstract

10614 Background: Identification of rare (>2-5) circulating tumor cells (CTC) in 7.5 ml blood by immunofluorescence assay (IFA) correlates with a poor prognosis in colon, breast, prostate and lung cancer. Changes in CTC count during treatment also predict the eventual patient progression and survival in these cancers. Existing assays do not detect melanoma CTC, however. In addition, isolation of viable CTC remains problematic. To overcome these limitations we attempted to develop novel melanoma CTC assays, using IFA and cell culture approaches. Methods: Blood samples were obtained from patients and controls following informed consent. The buffy coat (white cells + tumor) was isolated by Ficoll/Hypaque centrifugation, and split into 6 replicate cultures in proprietary TrueCells medium. After 21 days in culture, tumor colonies were counted, and stained for melanoma and leukocyte markers. Buffy coat cells from parallel blood samples were stained with a panel of CSPG4-specific mAb (a pan-melanoma marker) on ultraclean glass slides for analysis by immunofluorescence microscopy. Results: Blood samples were obtained from 16 melanoma patients, ages 28-87. Eight patients were men and 8 were women. CSPG4+ events (>2) were detected in 8/16 patients by IFA (range 0-52). In contrast, tumor cell colonies of >50 cells grew in 12 out of 16 patients with Stage 3 or 4 melanoma (range 0-1054), shown in Table. Cells isolated from CTC colonies produced melanin, stained for CSPG4 and other melanoma markers, but not for leukocyte markers. Control cultures grew no tumor colonies. Conclusions: Our pilot study shows that melanoma CTC can be identified by both IFA and cultured from blood in many patients with stage 3 or 4 melanoma. These CTC exhibited cytologic characteristics diagnostic of melanoma. The culture assay may represent a useful means of enumerating, isolating, and expanding viable melanoma CTC for further molecular study. [Table: see text]