General information
SALSA
® MLPA
® Probemixes P251/P252/P253 NB mix 1, 2 & 3 are research use only (RUO) assays for copy number determination of several chromosomal regions that frequently show copy number changes in neuroblastoma tumours.
Neuroblastoma (NB) is a relatively common paediatric cancer that usually occurs sporadically and frequently originates from one of the adrenal glands. NB is characterized by striking clinical heterogeneity, including cases that show spontaneous tumour regression. NB accounts up to 10% of all paediatric cancers. Several acquired genetic alterations such as amplification of the
MYCN oncogene, deletions of chromosome bands 1p36 and 11q23 and unbalanced gains of 17q regions have been well-characterized and show correlation with tumour behaviour, including response to treatment. For review please see e.g. Ambros et al. (2009) and Ahmed et al. (2017).
These products are not CE/FDA registered for use in diagnostic procedures. The SALSA® MLPA® technique is covered by US patent 6,955,901 and corresponding patents outside the US. The purchase of this product includes a license to use only this amount of product solely for the purchaser’s own use.
Probemix content
P251-C3 NB mix 1 contains 49 probes, including 36 probes for chromosomes 1, 3 and 11. P252-D1 NB mix 2 contains 49 probes, including 34 probes for chromosomes 2 (
MYCN region) and 17. P253-D2 NB mix 3 contains 47 probes, including 33 probes for chromosomes 4, 7, 9, 12 and 14. In addition, 13 reference probes are included in P251, 15 in P252 and 14 in P253 that target relatively copy number stable regions in various cancer types including NB. However, it should be noted that NB karyotypes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes. Partial probe sequences and the identity of the genes detected by the reference probes are available online (
www.mrcholland.com).
The P251 probemix contains probes for chromosomes 1, 3 and 11:
- 1p36: Deletion of the 1p36 region is present in 20-40% of NB patients and is often associated with
MYCN amplification in ∼70% of the cases (Huang and Weiss 2013). . A probe for tumour suppressor gene
CHD5 at 1p36 is included.
- 3p21-p22: 3p deletions are a hallmark of NB patients and appear to correlate with older age at diagnosis. This region contains various tumour suppressor genes, including
RASSF1 (Hoebeeck J et al. 2006; Van Roy et al. 2009;).
-11q: Deletions of the 11q arm, and in particular 11q23, are common in NB patients and are associated with a higher disease stage and poor prognosis (Mlakar V et al. 2017)
The P252 probemix contains probes for chromosomes 2 and 17:
- 2p24: Amplification of the proto-oncogene
MYCN is found in 20-30% of all NBs and it correlates with poor prognosis.
MYCN amplified tumours follow a very aggressive course and are associated with additional structural abnormalities — in particular with loss of 1p, 11q, and gain of 17q.
MYCN amplification is often used for identification of high-risk NB patients, and is also one of the most commonly mutated genes in retinoblastoma (RB) after the
RB1 gene.
MYCN is amplified in approximately 1-9 % of all RB tumors (Vempuluru VS et al. 2024). Additional probes for the nearby
NBAS,
DDX1 and
ALK genes are included.
- 2q33: Loss of 2q33 has been reported in NBs and has been associated with loss of expression of
CASP8 (Takita J et al. 2001).
- 17p: Gains of the 17p probes together with gains of the 17q probes would indicate complete chromosome 17 gains, in contrast to the frequent unbalanced 17q gains that are often associated with translocations. Three probes for
TP53 have been included although
TP53 mutations and deletions might be rare in NBs.
- 17q: 17q gain is present in more than 50% of NB cases and is associated with a poor outcome. Several studies showed that the most common frequent sites of 17q translocations are 1p and 11q. Whole chromosome 17 gain is typically seen in near-triploid tumours with favourable prognosis (Mlakar V et al. 2024).
Please note that triploidy of all chromosomes cannot be determined with these assays as only
relative gains or losses are detected.
The P253 probemix contains probes for chromosomes 4, 7, 9, 12 and 14:
- Partial copy number alterations of chromosomes 4, 7, 9, 12 and 14 have been described in NBs. Probes for
CDKN2A and
PTPRD on chromosome 9 have been included as
CDKN2A undergoes (often biallelic) deletion in many cancer types, and the
PTPRD gene has also been identified as a candidate tumour suppressor gene in NB (Carén H et al. 2008)
Each probemix contains nine quality control fragments generating amplification products between 64 and
105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at
www.mrcholland.com.
P252 competitor mix information
Samples with very high levels of MYCN amplification exhibit very high signals for the MYCN probes and low signals for other probes, making it difficult to analyse the latter. Therefore, the P252 probemix is shipped together with a vial of CM002 (P252 competitor mix). When a sample shows a very high level of MYCN amplification it can be retested with the competitor mix. This competitor mix contains oligonucleotides that can be included at the start of the MLPA reaction. Adding the competitors specifically reduces the signal of the eight MYCN region probes, making it possible to examine changes in other genes/chromosomal areas.
Instructions for use:
1. Denature 4 µl sample DNA by heating 5 minutes at 98°C.
2. Add 1.5 µl MLPA Buffer + 1.5 µl P252 probemix + 1 µl of P252 competitor mix.
3. Continue with the MLPA protocol starting with one minute incubation at 95°C and 16 hour incubation at 60°C followed by the ligation and PCR steps.