title: Molecular detection of tyrosinase transcripts in peripheral blood from patients with malignant melanoma: correlation of PCR sensitivity threshold with clinical and pathologic disease characteristics.
abstract: BACKGROUND
  Positive molecular detection of tyrosinase transcripts (TYR mRNA) in RNA extracts of peripheral blood (PB) samples from patients with malignant melanoma provides evidence of disease dissemination.
  
  
  METHODS
  Total RNA extracted from PB was quantified and subjected to RT-PCR under ultra-sensitive and reduced-sensitivity PCR conditions using SSRT-II. Positive TYR mRNA detection in 78 melanoma patients and 40 healthy volunteers was correlated with clinical stage, Breslow's evaluation of tumor thickness, Clark's assessment of tumor invasion, the location of the primary tumor site, and tumor histology. The assay sensitivity was evaluated by spiking PB with the melanoma cell line SK-MEL-28.
  
  
  RESULTS
  Using ultra-sensitive PCR conditions, eight out of 40 RNA (20%) samples from healthy volunteers and 50 out of 78 RNA (64.1%) samples from melanoma patients tested positive. Using reduced-sensitivity PCR conditions, we found only two positives in 40 RNA samples from healthy subjects and 20 positives in 78 RNA samples (25.6%) from melanoma patients. Only positive PCR samples for the reduced-sensitivity PCR assay correlated significantly with stage IV (metastatic) disease (p=0.0395). There was no significant correlation between positive TYR mRNA samples for either PCR condition (ultra-sensitive and reduced-sensitivity) with Breslow's classification of tumor thickness, Clark's assessment of tumor invasion, location of the primary tumor site, and type of tumor histology.
  
  
  CONCLUSIONS
  We conclude that reduced-sensitivity rather than ultra-sensitive PCR conditions correlate with clinical stage in melanoma patients.
authors.affiliation: Department of Experimental Physiology, Medical School, University of Athens, Micras Asias 75, Goudi, 11527 Athens, Greece.
authors.forename: Georgios
authors.initials: G
authors.lastname: Mitropapas
authors.forename: Andrianos
authors.initials: A
authors.lastname: Nezos
authors.forename: Antonis
authors.initials: A
authors.lastname: Halapas
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Pissimissis
authors.forename: Peter
authors.initials: P
authors.lastname: Lembessis
authors.forename: Antigone
authors.initials: A
authors.lastname: Sourla
authors.forename: Pericles
authors.initials: P
authors.lastname: Vassilopoulos
authors.forename: Michael
authors.initials: M
authors.lastname: Koutsilieris
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008545:Melanoma / Q000097:blood / Q000235:genetics / Q000473:pathology*; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009367:Neoplasm Staging; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D014442:Monophenol Monooxygenase