Potential application of ELAVL4 real-time quantitative reverse transcription-PCR for detection of disseminated neuroblastoma cells.
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.
Swerts
Katrien
K
Department of Pediatric Hematology and Oncology, Ghent University Hospital, Ghent, Belgium. Katrien.Swerts@UGent.be
De Moerloose
Barbara
B
Dhooge
Catharina
C
Vandesompele
Jo
J
Hoyoux
Claire
C
Beiske
Klaus
K
Benoit
Yves
Y
Laureys
Geneviève
G
Philippé
Jan
J
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
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