title: Quantitative detection of circulating tumor cells in cutaneous and ocular melanoma and quality assessment by real-time reverse transcriptase-polymerase chain reaction.
abstract: OBJECTIVE
  Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells.
  
  
  METHODS
  We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n = 18), peripheral blood samples from healthy donors (n = 21), and patients with cutaneous (n = 122) and uveal (n = 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples.
  
  
  RESULTS
  Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/ microl porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples).
  
  
  CONCLUSIONS
  Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.
authors.affiliation: Department of Medicine III, University Hospital Benjamin Franklin, Free University Berlin, Berlin, Germany. ulrich.keilholz@medizin.fu-berlin.de
authors.forename: Ulrich
authors.initials: U
authors.lastname: Keilholz
authors.forename: Petra
authors.initials: P
authors.lastname: Goldin-Lang
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Bechrakis
authors.forename: Nicole
authors.initials: N
authors.lastname: Max
authors.forename: Anne
authors.initials: A
authors.lastname: Letsch
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmittel
authors.forename: Carmen
authors.initials: C
authors.lastname: Scheibenbogen
authors.forename: Karin
authors.initials: K
authors.lastname: Heufelder
authors.forename: Alexander
authors.initials: A
authors.lastname: Eggermont
authors.forename: Eckhard
authors.initials: E
authors.lastname: Thiel
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005134:Eye Neoplasms / Q000097:blood / Q000235:genetics / Q000473:pathology*; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D058965:MART-1 Antigen; D008545:Melanoma / Q000097:blood / Q000235:genetics / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000097:blood / Q000235:genetics; D014442:Monophenol Monooxygenase / Q000235:genetics; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic; D058951:gp100 Melanoma Antigen
chemical_list: D000951:Antigens, Neoplasm; D017931:DNA Primers; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D058951:gp100 Melanoma Antigen; D014442:Monophenol Monooxygenase; D011163:Hydroxymethylbilane Synthase