Standardisation of operating procedures for the detection of minimal disease by QRT-PCR in children with neuroblastoma: Quality assurance on behalf of SIOPEN-R-NET

Consensus criteria for sensitive detection of minimal neuroblastoma cells in bone marrow, blood and stem cell preparations by immunocytology and QRT-PCR: recommendations by the International Neuroblastoma Risk Group Task Force

“…However the clinical benefit of QRT-PCR for detection of disease will most likely come from the sensitivity and specificity with which low-level disease (o10%) is detected. To minimise intra-and inter-assay variability across different laboratories, SOPs for the optimal analysis and reporting of MD detected by QRT-PCR for TH mRNA in haemopoietic compartments have been established (Viprey et al, 2007). These SOPs have been adopted by the INRG Metastatic Disease Committee and are summarised below.…”

“…Despite this literature the clinical utility of RT -PCR for TH mRNA remains unclear, reflecting the small number of patients studied, absence of quality control across studies, lack of uniform methodology for sample collection, processing and inconsistency in reporting (Riley et al, 2003). Therefore the clinical utility of RT -PCR for TH mRNA is currently being evaluated in a large prospective clinical trial (HR-NBL1/ ESIOP; www.siopen-r-net.org), according to quality-controlled SOPs (Viprey et al, 2007).…”

“…cDNA is synthesised from 10 ml of RNA (400 ng) for 1 h at 371C, reverse transcriptase enzyme is inactivated by heating at 951C for 5 min and amplified using the TaqMan assay as previously described (Viprey et al, 2007). Each sample is amplified in triplicate (3 Â cDNA from 100 ng RNA) for TH mRNA and once (1 Â 80 ng) for the housekeeping gene b2M.…”

“…This is particularly important for defining the clinically relevant level of MD and MRD at diagnosis and during disease course respectively. For accurate reporting the selection of an optimal reference mRNA against which the test mRNA(s) can be normalised is essential (Beillard et al, 2003;Viprey et al, 2007). b-2 microglobulin (b2M) is frequently selected as the standard housekeeping gene to report expression of MD in BM, PB and/or PBSC as it is stably expressed in normal haematopoietic cells (Livak and Schmittgen, 2001;Beillard et al, 2003).…”

Recurrent Neuroblastoma

“…For IC, both the SIOPEN Bone Marrow Committee and the INRG Task Force have recommended disialoganglioside (G D2 ) as a target cell surface antigen and have developed standardized protocol for IC (Beiske et al, 2009;Swerts et al, 2005). Similarly, standard operating procedures have been developed by the SIOPEN Research Network and INRG Task Force for QRT-PCR, using tyrosine hydroxylase as target mRNA (Beiske et al, 2009;Viprey et al, 2007). The use of multiple molecular markers in QRT-PCR to increase the sensitivity and specificity of MRD detection has also been investigated (I.Y.…”