{'fraction_non_alphanumeric': 0.03548477228276055, 'fraction_numerical': 0.0131321598211791, 'mean_word_length': 5.45045045045045, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
A novel method to determine circulating malignant cells in the peripheral blood of melanoma patients

IntroductionIn the last three decades several attempts have been made to detect circulating tumor cells in the blood of cancer patients in order to obtain valuable information on prognosis, recurrence after therapy, or response to treatment. However, consolidated method able to provide satisfactory answers to these problems in melanoma patients are not presently available. Aim of the studyTo test novel strategies for evaluating circulating melanoma cells (CMC), using an experimental approach based on “simulation design”, in which cultured melanoma cells are added to peripheral blood of normal donors. MethodsTwo long-term cultured melanoma lines (M10 and SK-Mel-28) and 5 primary cultures of melanoma cells obtained from patients bearing metastatic disease were used. CMC separation phase: graded numbers of melanoma cells, prelabeled in vitro with 51Cr, were added to 5–10 ml of heparinized blood, freshly collected from healthy donors. Each experiment was run in parallel with the same number of non-radioactive melanoma cells. Thereafter, mononuclear cells (MNC) were separated by centrifugation on ficoll-hypaque gradient, and “artificial” CMC were isolated using: (a) direct “capture” of melanoma cells (DC) by immunomagnetic beads, or (b) enrichment of melanoma cells by negative selection (ENS). The extent of recovery of prelabeled cells was calculated on the basis of radioactivity of recovered cell fractions. CMC recognition phase: non-prelabeled melanoma cells isolated by means of DC or ENS method, were recognized by their telomerase activity (TLMA) tested according to telomeric repeat amplification protocol (TRAP), either adopting the classic radioactive procedure, or the non-radioactive ELISA assay (Roche). This assay was adopted since it was considered to be extremely sensitive and relatively selective for neoplastic cells. ResultsPreliminary experiments with DC methods, using immunomagnetic beads coated with monoclonal antibodies (mAbs) recognizing melanocytes or melanoma cells did not provide sufficiently high recovery rates of prelabeled cells. Therefore, all subsequent studies were based on ENS-mediated CMC separation technique, in agreement with a previous study (Georgieva J. et al: Melanoma Res. 12: 309–317, 2002). In particular, lymphocytes present in MNC separated from blood were captured with graded amounts of immunomagnetic beads coated with anti-CD45 mAb (Oxoid). The supernatant, containing the non-captured cells (i.e. the majority of melanoma cells that are CD45-negative) was centrifuged and the pellet used for radioactivity count (to test the overall melanoma cell recovery) and TLMA determination (to evaluate sensitivity of the assay). The results of these studies showed that: (a) recovery of prelabeled cells by means of ficoll-hypaque gradient centrifugation (i.e. after the first separation step) did not exceed 75%; (b) further recovery of prelabeled cells after negative selection operated by adequate amounts (i.e. 400–500 μl) of anti-CD45 immunomagnetic beads did not exceed 85%. Therefore, the overall recovery of prelabeled cells added to blood did not exceed 60–65%. (c) TLMA evaluated on melanoma cells recovered in parallel experiments performed with non-prelabeled cells, showed that this technique was able to detect a number of recovered melanoma cells ranging from 50 to 200 cells, depending from the intrinsic TLMA of melanoma cells.Supported by PNR of the Ministry of Education and Research (Italy).