Detection of Neuroblastoma Cells During Clinical Follow Up: Advanced Flow Cytometry and RT-PCR for Tyrosine Hydroxylase Using Both Conventional and Real-Time PCR

Abstract

Purpose:

Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary.

Methods:

We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC).

Results:

Cohen’s kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression.

Conclusion:

TH RT-qPCR alone is limited for detection of NB cells because of “false positives” in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.

Zusammenfassung

Hintergrund:

Die Real-time-Reverse-Transkriptase PCR (RT-qPCR) oder konventionelle RT-cPCR für Tyrosinhydroxylase (TH) dient zum Nachweis von Neuroblastomzellen (NB). Die TH-Expression in Normalgewebe erfordert weitere diagnostische Strategien.

Methoden:

Wir untersuchten 857 Proben von 65 NB-Patienten mit RT-cPCR für TH und verglichen die Ergebnissen mit 666 Proben aus der RT-qPCR. Als Kontrolle wurden 84 Proben von Patienten mit anderen Diagnosen und 354 Proben gesunder Spender geprüft. 132 Proben wurden mit 5-Farb-Flowzytometrie hinsichtlich des Vorhandensein von NB-Zellen ausgewertet.

Ergebnissse:

Die Cohen’s-Kappa-Koeffizienten zeigten eine beachtliche Übereinstimmung sowohl im Vergleich der RT-cPCR vs. RT-qPCR als auch der RT-cPCR vs. FC und eine mittelmäßige Übereinstimmung der RT-qPCR vs. FC. FC-Panel bieten eine weitere diagnostische Strategie, die NB-Zellen als CD45negCD9+CD81+CD56+ch14:18+GD2+ charakterisiert mit gelegentlicher Expression von CD57, CD138, CD166 und einer Nachweisgrenze von 0,002%.

Schlussfolgerung:

Aufgrund falsch positiver Ergebnisse bei Proben von Patienten mit anderen Erkrankungen ist die RT-qPCR allein nur begrenzt zum Nachweis von NB-Zellen geeignet. Die erweiterte FC als komplementäre Methode zur Detektion von NB-Zellen ist im größeren Kollektiv zu prüfen.

Key words

neuroblastoma - minimal residual disease - flow cytometry - RT-PCR for TH

Schlüsselwörter

Neuroblastom - minimale Resterkrankung - Durchflusszytometrie - RT-PCR für TH

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