A Multilocus Technique for Risk Evaluation of Patients with Neuroblastoma

Abstract

Purpose: Precise and comprehensive analysis of neuroblastoma genetics is essential for accurate risk evaluation and only pangenomic/multilocus approaches fulfill the present-day requirements. We present the establishment and validation of the PCR-based multiplex ligation-dependent probe amplification (MLPA) technique for neuroblastoma.Experimental Design: A neuroblastoma-specific MLPA kit was designed by the SIOP Europe Neuroblastoma Biology Committee in cooperation with MRC-Holland. The contained target sequences cover 19 chromosomal arms and reference loci. Validation was performed by single locus and pangenomic techniques (n ¼ 174). Dilution experiments for determination of minimal tumor cell percentage were performed and testing of reproducibility was checked by interlaboratory testing (n ¼ 15). Further 156 neuroblastomas were used for establishing the amplification cutoff level.Results: The MLPA technique was tested in 310 neuroblastomas and 8 neuroblastoma cell lines (including validation and amplification cutoff level testing). Intertechnique validation showed a high concordance rate (99.5%). Interlaboratory MLPA testing (k ¼ 0.95, P < 0.01) revealed 7 discrepant of 1,490 results (0.5%). Validation by pangenomic techniques showed a single discordance of 190 consensus results (0.5%). The test results led to formulation of interpretation standards and to a kit revision. The minimal tumor cell percentage was fixed at 60%.Conclusions: The recently designed neuroblastoma-specific MLPA kit covers all chromosomal regions demanded by the International Neuroblastoma Risk Group for therapy stratification and includes all hitherto described genetic loci of prognostic interest for future studies and can be modified or extended at any time. Moreover, the technique is cost effective, reliable, and robust with a high interlaboratory and intertechnique concordance.

Genomic Copy Number Profiling Using Circulating Free Tumor DNA Highlights Heterogeneity in Neuroblastoma

“…In neuroblastoma, it has been shown that the overall tumor genomic profile determined by copy number analysis of tumor material is of prognostic impact, and genomic copy number profiling using material from tumor cells is considered as a reference for obtaining a tumor genomic profile at the time of diagnosis (11,13,14). We have now undertaken a study of 70 plasma samples obtained at diagnosis to determine the feasibility of using cfDNA isolated from plasma for the study of an overall genomic profile.…”

“…Current treatment strategies take into account the genomic copy number profile both in patients with low-risk neuroblastoma, as well as in certain patients with high-risk neuroblastoma, aged <18 months (11,12). A genomic copy number profile is routinely assessed on diagnostic tumor tissue, most frequently using pangenomic techniques, such as MLPA, array comparative genomic hybridization (aCGH), or SNPa (11,13,14). However, in some instances, biopsies from a primary or metastatic tumor site are not available, for instance, in severely ill young infants with disseminated disease, whereas in older children biopsies might harbor only a low percentage of tumor cells, thus impacting on molecular genetic analysis.…”

Changes in MYCN expression in human neuroblastoma cell lines following cisplatin treatment may not be related to MYCN copy numbers

“…Notably, observations based on metaphase FISH analyses of SK-N-AS CDDP cells (revealing cells with 2p gain, sporadic tetrasomy or monosomy) have not been confirmed by MLPA analysis, which did not confirm any significant 2p change in both SK-N-AS or SK-N-AS CDDP cell lines. This discrepancy may be due to the nature of the two techniques: FISH may be more subjective due to the visual evaluation, while capable of taking into account the heterogeneity in the cell population, wherease MLPA, an accurate and sensitive technique, analyzes whole genomic material irrespective of the situation in particular cells (33). Our data suggest the need to analyze amplification of the genetic material by two independent methods in order to avoid false interpretations.…”

Ten Challenges in the Management of Neuroblastoma

“…Their use in future science group Ten challenges in the management of neuroblastoma Review the future stratification of patients in the clinical trial setting is being discussed. Multiplex ligation-dependent probe amplification is now being used to select patients with segmental chromosomal abnormalities rapidly [20]. These correlate with the segmental chromosomal abnormalities identified by array comparative genomic hybridization and shown to be of prognostic significance in the <18-month-old age range [21].…”