Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used as described13 (link),29 (link),30 (link) to assess MRD in heparinized BM aspirates pooled from four sites (2–2.5 ml/site, from bilateral posterior and anterior iliac crests). The MRD marker panel included cyclin D1 (CCND1), GD2 synthase (B4GALNT1), ISL LIM homeobox 1 (ISL1) and paired-like homeobox 2b (PHOX2B). β2 microglobulin (β2M) was used as the endogenous control, and NB cell line NMB7 as the positive control. Each sample was quantified using the comparative CT method as fold-difference relative to NMB7. All gene expression assays were from Applied Biosystems: CCND1: Hs00277039_m1; B4GALNT1: Hs00155195_m1; ISL1: Hs00158126_m1; PHOX2B: Hs00243679_m1; β2M: 4326319E. For each marker, positivity was defined as greater than the upper limit of normal. All samples were run in duplicates. MRD panel positivity was defined as any one of 4 markers being positive, and negativity as all 4 markers being negative.
Protocol detail
Quantifying Minimal Residual Disease in Neuroblastoma
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Assays
B4galnt1
Cell line
Cyclin d1
Gd2 synthase
Gene expression
Homeobox
Iliac crests
Reverse transcription polymerase chain reaction
Corresponding Organization : Memorial Sloan Kettering Cancer Center
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Variable analysis
independent variables
- None explicitly mentioned
- Minimal Residual Disease (MRD) levels
- Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) protocol as described in references 13, 29, and 30
- Heparinized bone marrow (BM) aspirates pooled from four sites (2–2.5 ml/site, from bilateral posterior and anterior iliac crests)
- MRD marker panel: cyclin D1 (CCND1), GD2 synthase (B4GALNT1), ISL LIM homeobox 1 (ISL1) and paired-like homeobox 2b (PHOX2B)
- β2 microglobulin (β2M) as the endogenous control
- NB cell line NMB7 as the positive control
- NB cell line NMB7
- None explicitly mentioned
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