title: Potential application of ELAVL4 real-time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells.
abstract: BACKGROUND
  Reliable detection of neuroblastoma cells in bone marrow (BM) is critical because BM involvement influences staging, risk assessment, and evaluation of therapeutic response in neuroblastoma patients. Standard cytomorphologic examination of BM aspirates is sensitive enough to detect single tumor cells. Consequently, more sensitive and specific detection methods are indispensable.
  
  
  METHODS
  We used real-time quantitative reverse transcription-PCR (QPCR) of the tyrosine hydroxylase (TH), GD2 synthetase (GALGT), and embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) genes to detect disseminated neuroblastoma cells. We assessed assay sensitivity by addition experiments and then analyzed 97 neuroblastic tumor, BM, peripheral blood (PB), or peripheral blood stem cell (PBSC) samples from 30 patients. The QPCR results were compared with those of a standardized immunocytochemical assay.
  
  
  RESULTS
  The molecular markers were highly expressed in all evaluated tumor samples. In addition, 32%, 11%, and 38% of all BM, PB, and PBSC samples scored positive for TH, GALGT, or ELAVL4, respectively. The TH and ELAVL4 assays could detect 1 neuroblastoma cell in 10(6) mononuclear cells. By contrast, the GALGT QPCR assay could detect 1 neuroblastoma cell in 10(4) mononuclear cells. We assessed the potential prognostic value of TH, GALGT, and ELAVL4 QPCR by analyzing subsequent samples from 3 patients with stage 4 disease. Preliminary results indicated that persistence of high ELAVL4 expression has prognostic value.
  
  
  CONCLUSIONS
  ELAVL4 QPCR can be used to detect residual neuroblastoma cells in clinical samples. However, combination of several molecular markers and screening techniques should be considered to ensure reliable detection of rare neuroblastoma cells.
authors.affiliation: Department of Pediatric Hematology and Oncology, Ghent University Hospital, Ghent, Belgium. Katrien.Swerts@UGent.be
authors.forename: Katrien
authors.initials: K
authors.lastname: Swerts
authors.forename: Barbara
authors.initials: B
authors.lastname: De Moerloose
authors.forename: Catharina
authors.initials: C
authors.lastname: Dhooge
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.forename: Claire
authors.initials: C
authors.lastname: Hoyoux
authors.forename: Klaus
authors.initials: K
authors.lastname: Beiske
authors.forename: Yves
authors.initials: Y
authors.lastname: Benoit
authors.forename: Geneviève
authors.initials: G
authors.lastname: Laureys
authors.forename: Jan
authors.initials: J
authors.lastname: Philippé
mesh_terms: D000293:Adolescent; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D001853:Bone Marrow / Q000737:chemistry / Q000473:pathology; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D051959:ELAV Proteins / Q000032:analysis* / Q000235:genetics; D000067779:ELAV-Like Protein 4; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000235:genetics; D018365:Neoplasm, Residual; D009360:Neoplastic Cells, Circulating / Q000737:chemistry / Q000473:pathology*; D009447:Neuroblastoma / Q000737:chemistry / Q000175:diagnosis / Q000473:pathology*; D011379:Prognosis; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013234:Stem Cells / Q000378:metabolism / Q000473:pathology; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; C498010:ELAVL4 protein, human; D012333:RNA, Messenger; D014446:Tyrosine 3-Monooxygenase; D017350:N-Acetylgalactosaminyltransferases; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase