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      Prognostic significance of the sequential detection of circulating melanoma cells by RT–PCR in high-risk melanoma patients receiving adjuvant interferon

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          Abstract

          The purpose of this study was to address the prognostic significance of circulating melanoma cells by reverse transcriptase-polymerase chain reaction in the peripheral blood of stage IIB and III melanoma patients on high-dose adjuvant interferon at multiple sequential time points from initiation of treatment. Tyrosinase mRNA in peripheral blood from these patients was assayed by reverse transcriptase polymerase chain reaction prior to initiation of adjuvant interferon, at completion of 1 month of intravenous interferon and at 3 monthly intervals until progression. Four hundred and eighteen blood samples from 60 melanoma patients were analysed. The median follow-up time calculated from the time of inclusion in the study was 23 months (range 2–38 months). Tyrosinase mRNA in blood was detected in 42 (70%) of 60 patients: 16 (76%) of 21 stage IIB patients and 26 (66%) of 39 stage III patients. The presence of tyrosinase mRNA in blood was correlated with a shorter disease-free survival ( P : 0.03) and in multivariante analysis was an indepent prognostic factor for relapse. Patients who seroconverted to a negative reverse-transcriptase-polymerase chain reaction after induction treatment had a significantly lower probability of recurrence. The presence of circulating melanoma cells is a marker of a high relapse risk and shorter disease-free survival whether detected postoperatively or during follow-up. Tyrosinase mRNA amplification by reverse-transcriptase-polymerase chain reaction may be a useful tool for monitoring the efficacy of adjuvant treatment in stage IIB and III melanoma patients.

          British Journal of Cancer (2002) 87, 181–186. doi: 10.1038/sj.bjc.6600419 www.bjcancer.com

          © 2002 Cancer Research UK

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction.

            Only small numbers of cells from solid tumours are needed for haematogenous metastasis. Detection is difficult because existing techniques are not sensitive enough. We have used reverse transcriptase to make complementary DNA from peripheral blood messenger RNA, and the polymerase chain reaction (PCR) to amplify cDNA specific for a gene actively transcribed only in the tumour tissue type. We prepared cDNA from peripheral blood of seven patients with malignant melanoma, four patients with other metastatic cancers, and four healthy subjects, as well as from several melanoma-derived cell lines. PCR was used to amplify the gene for tyrosinase, a tissue-specific gene in melanocytes. Since normal melanocytes are not thought to circulate in peripheral blood, detection of tyrosinase transcription in peripheral blood should indicate the presence of circulating cancer cells. The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood. Blood samples from four of the seven patients with malignant melanoma gave positive results, whereas all eight control subjects gave negative results. This method does not depend on the characterisation of cancer-specific genetic abnormalities and can be applied to any cancer for which tissue-specific genes can be identified, including epithelial cancers. It could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment.
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              Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus.

              Screening of a lambda gt11 human melanocyte cDNA library with antibodies against hamster tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were from related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase.
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                Author and article information

                Journal
                Br J Cancer
                British Journal of Cancer
                Nature Publishing Group
                0007-0920
                1532-1827
                02 July 2002
                15 July 2002
                : 87
                : 2
                : 181-186
                Affiliations
                [1 ]Research Laboratory of 1st Department of Medicine, University of Athens; Aghiou Thoma 17, Goudi, Athens 11 527, Greece
                [2 ]Hellenic Cooperative Oncology Group (HeCOG), Data office, Laskaridou 1, Athens 11 524, Greece
                Author notes
                [* ]Author for correspondence: hgogas@ 123456hol.gr
                Article
                6600419
                10.1038/sj.bjc.6600419
                2376114
                12107840
                2fe98bb7-b02c-49b2-9559-a51cd2d7d14f
                Copyright 2002, Cancer Research UK
                History
                : 03 December 2001
                : 08 April 2002
                : 29 April 2002
                Categories
                Molecular and Cellular Pathology

                Oncology & Radiotherapy
                rt–pcr,tyrosinase mrna,interferon,melanoma
                Oncology & Radiotherapy
                rt–pcr, tyrosinase mrna, interferon, melanoma

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