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

Abstract

Disseminating disease is a predictive and prognostic indicator of poor outcome in children with neuroblastoma. Its accurate and sensitive assessment can facilitate optimal treatment decisions. The International Neuroblastoma Risk Group (INRG) Task Force has defined standardised methods for the determination of minimal disease (MD) by immunocytology (IC) and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) using disialoganglioside G(D2) and tyrosine hydroxylase mRNA respectively. The INRG standard operating procedures (SOPs) define methods for collecting, processing and evaluating bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell harvest by IC and QRT-PCR. Sampling PB and BM is recommended at diagnosis, before and after myeloablative therapy and at the end of treatment. Peripheral blood stem cell products should be analysed at the time of harvest. Performing MD detection according to INRG SOPs will enable laboratories throughout the world to compare their results and thus facilitate quality-controlled multi-centre prospective trials to assess the clinical significance of MD and minimal residual disease in heterogeneous patient groups.

Figure 1

Consensus guidelines for sampling bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell (PBSC) harvest for immunocytology (IC) and reverse transcriptase (RT)–PCR.

Figure 2

Immunocytological analysis. Consensus guidelines for cell preparation and immunocytochemical staining. Abbreviations: r.p.m., rounds per min; PBS, phosphate-buffered saline; BSA, bovine serum albumin; APAAP, alkaline phosphatase anti-alkaline phosphatase.

Figure 3

Immunocytological analysis. Consensus guidelines for light microscopic evaluation, cell counting and reporting of results.

Figure 4

(A–L) Immunostaining of cytospins containing mononuclear cells from BM aspirates pulled from children with NB stage M. The red label shows binding of monoclonal anti-G_D2 antibody 14.G2a visualised by a commercial alkaline phosphatase anti-alkaline phosphatase kit and Fuchsin+ substrate (Swerts et al, 2005). All images are taken with a × 60 dry lens and reproduced with identical magnification to enable comparison of different cell types by size. A and B show the mandatory cytomorphological and immunological criteria of neuroblasts, that is high nuclear/cytoplasmic ratio and strong G_D2 membrane expression. A granular appearance of the chromatin is frequently, but not always recognised. Neuroblasts are usually larger than small lymphocytes (arrows). If all criteria are fulfilled, even single neuroblasts can be identified (B). Reliable recognition of cytomorphological and immunological criteria depends on paraformaldehyde fixation. (C) Typical clump formation of neuroblasts with moulding nuclei. Some tumour cells may show increased amount of cytoplasm (arrow), but the nuclear/cytoplasmic ratio should not decline below 2. Note adjacent G_D2-negative lymphocytes. Cells that fulfil the criteria as detailed in A–C are classified as criteria-positive cells (CPC). (D–F) Shedding of G_D2 from a clump (D) or a single neuroblast (arrow in E) on adjacent haematopoietic cells creates the impression of an almost complete (arrow in D) or partial membrane staining. G_D2 may even shed from fibrillary acellular material, for example fragment of neuropile (arrow in F) on to normal cells. Note: no neuroblasts are identified in F. (G–I) Not convincingly interpretable cells (NCICs). Two of the cells in G (arrows) do not display a complete membrane staining. However, their nuclear size, morphology and clump formation strongly favour a neuroblastic origin. In H, form and size of the nucleus suggest a neuroblast, but the cell membrane is widely avoided of G_D2. Also the cell in I is incompletely stained and in addition somewhat smaller than typical neuroblasts. The true nature of these cells can only be revealed by means of ancillary methods (eg FISH, single-cell PCR). Their potential clinical significance remains to be established. (J–L) Macrophages with ingested G_D2 are often found after chemotherapy and must not be interpreted as tumour cells. Hallmarks of macrophages are a very low nuclear/cytoplasmic ratio (usually <1), a granular compartmentalisation of the antigenic material in vesicles and a negative membrane stain (arrows in J–L), although the label may even occupy the whole cytoplasm (L). The colour quality of ingested G_D2 is usually not purple red as in neuroblasts (see A–E), but rather brownish due to increased iron storage after blood transfusions.

Figure 5

RNA is extracted and processed for detection of mRNA by QRT-PCR according to standard operating procedures.