Table 1.

Nomenclature and threshold levels for MLPA results, partly published in Ambros and colleagues (2)

TermsDescriptionComments
Normal statusBalanced ratio between the majority of signalsa of both chromosomal armsIn the case of a balanced ratio, a uniparental isodisomyb with a complete or partial LOH of loci located on the investigated chromosome cannot be excluded
Caveat: flat profiles most likely reflect a too small tumor cell content and are designated as “no result”
Segmental chromosome aberration
 LossUnbalanced ratio between the signals of the chromosomal region of interest (decrease of the mean value of at least 2 adjacent probes of at least 0.25) and the reference signals (at least 2 adjacent probes in direct vicinity)This result could correspond to a FISH deletion, which reflects an LOH, or a FISH imbalance, which does not necessarily indicate an LOH
 GainUnbalanced ratio (low signal excess, between at least 0.25 and 1.0) between the signals of the chromosomal region of interest (at least 2 adjacent probes) and the reference signals (at least 2 adjacent probes in direct vicinity) of the chromosomal region of interestIn case of chromosome 2p gain, a type 1 gain is discriminated from a type 2 gain (see also Discussion); the latter is suspicious for either MYCN amplification in a minority of tumor cells (intratumoral heterogeneity) or MYCN amplification in samples with very low tumor cell numbers (e.g., after therapy), resulting in flat curves except for MYCN (and possibly co-amplified genes); clarification by I-FISH is highly recommended
AmplificationUnbalanced ratio (high signal excess, at least 12 × 0.25, i.e., a ratio of 3) between the signals of one or more genes (at least 2 adjacent probes) and all other probes located on the same chromosomeSignal excess ratios between 1.0 and 3 are suspicious for the presence of small numbers of MYCN amplified tumor cells-–either because of low tumor cell content (flat MLPA profile) or due to heterogeneous MYCN amplification and have to be clarified by I-FISH
Borderline mean valuesDifferences of mean values between 0.2 and 0.24 are regarded as borderline. MLPA should be repeated or other techniques be used for clarification. A homogenous increase or decrease of several adjacent probes not reaching the borderline value of 0.2 to 0.24 can be regarded as suspicious for a SCA and should be clarified by another technique.
Outlier probeOnly 1 probe shows a more than 0.25-fold increased or decreased value-–not taken into account in the calculation of mean values of other probes located on the concerned chromosome. FISH, aCGH, and/or SNP array can be used for clarification. In case a chromosomal arm is only covered by 3 probes, it cannot be evaluated (in the current kit, the 3q, 7p, 12p and 12q arms are concerned).
No resultShould be specified: unclear or not interpretable (e.g., flat profile or too much fluctuation, mostly due to inadequate tumor cell content or degraded DNA, respectively)
No tumor; not done
  • aSignal intensity is visualized in the graphic representation of the MLPA results as height of the bars.

  • bA uniparental isodisomy means that both chromosomes or parts thereof are derived from one parent. Thus, the 2 chromosomes are identical. However, uniparental isodisomies are rare in NB tumors (they are found in 1%; G.S. and J.C., unpublished).