SALSA^® digitalMLPA™ Probemix D006-A1 Multiple Myeloma is a research use only (RUO) assay for the detection of deletions, gains or amplifications of genes and chromosomal regions mentioned in Table 2 of the product description that are recurrently altered in multiple myeloma, such as 1p, 1q, 13q and 17p, as well as for the detection of BRAF p.V600E point mutation.

Multiple myeloma (MM) is a clonal B-cell disorder characterised by malignant proliferation of monoclonal plasma cells. MM cases present with a common histological and morphological diagnosis, however simultaneously displaying enormous genetic and molecular complexity as well as marked variations in clinical characteristics and patient survival. Recent progress in molecular cytogenetics has improved the understanding of pathogenesis of MM and also provided reasoning for molecular sub-classification of MM. Genetic alterations in MM are well characterised and include gross chromosomal rearrangements such as fusion genes, hyper-/hypodiploidy and also focal deletions. This probemix is designed to detect the majority of the primary and secondary CNAs in MM.

The image below shows key target genes and regions for this probemix. Click the image to enlarge.

Use the calculator below to get an indication of the number of samples that can be included in a sequencing run. Click here if you are having issues loading or using the calculator, or if you would like more information about the calculation.

Tumour tissues exhibit significant heterogeneity, characterised by varying tumour cell percentages (presence of non-tumour cells) and often subclonal cell expansions as a result of cancer evolution. When extracting DNA from a tumour tissue sample, this includes some DNA from non-tumour cells and genetic information from possible different (sub)clones of the tumour.

digitalMLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. However, if the percentage of tumour cells is low, gains or losses of genomic regions or genes may not be detected. In addition, the subclonality of the aberration affects the inter ratio of the corresponding probe.

In case of a deletion that is present in a lower percentage of tumour cells and/or subclonal, the inter ratio may be higher than expected. For example, a monoallelic deletion in a sample with 60% tumour cell content (or a monoallelic deletion present in 60% of the tumour cells) will result in an inter ratio around 0.70. However, the same inter ratio of 0.70 will also be found in a sample with a biallelic deletion and a tumour cell percentage of 30% (or a subclone comprising 30% of all tumour cells) (this example can be found in bold in Table 1). The digitalMLPA technique cannot discriminate between these two scenarios.

Theoretical inter ratios obtained with digitalMLPA with a specific percentage of cells carrying the aberration can be found below. Estimating the percentage of cells carrying the aberration (tumour cell percentage and/or possible subclonality) could help facilitate the interpretation. As can be appreciated in this table, certain inter ratios can be interpreted in various ways, depending on the tumour cell percentage and/or subclonality.

Table 1: Theoretical expected median inter ratios for aberrations present at different percentages.

As a real-life example of the results of digitalMLPA experiments with different percentages of cells carrying the aberration, DNA from a set of eight Coriell samples and tumour cell lines was diluted with different percentages of "healthy" DNA (without detectable CNAs with SALSA digitalMLPA Probemix D006 Multiple Myeloma). A table with inter ratios observed in this sample set for a specific percentage of cells carrying the deviation (CNA) can be found below.

Table 2: Median inter ratios observed for aberrations present at different percentages when tested with SALSA digitalMLPA Probemix D006 Multiple Myeloma version A1. Please carefully take the variation of the inter ratios within your experiment into account.

* Inter ratios for amplifications (gain of >2 copies) are expected to follow the same trend, with lower values when less cells carry the aberration).

As can be observed in the table above, with lower percentages of cells carrying the aberration, inter ratios of samples with CNAs overlap with the expected normal range of inter ratios (0.85–1.15). We conclude the following: the minimum percentage of tumour cells required for reliable analysis is 30%, which is in line with previously published data (Al Zaabi et al. 2010, Coll-Mulet et al. 2008, Benard-Slagter et al. 2017). We recommend using tumour samples with at least 50% tumour cell content to minimize the variation in tumour cell estimation, and to allow robust data-analysis and detection of potential subclonal aberrations.

SALSA digitalMLPA Probemix D006 Multiple Myeloma is for research use only (RUO) in all territories.

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

See this support article for commercially available positive samples that have been tested with this product.

Key related products

• P425 Multiple Myeloma: Targets genes and regions associated with multiple myeloma: 1p32-p12, 1q21-q23, 5q31, chr. 9, 12p13, 13q14-q22 (RB1-DLEU2-DIS3), 14q32 (TRAF3), chr. 15, 16q12 (CYLD), 16q23 (WWOX), and 17p13 (TP53).
• P056 TP53: Targets each exon of TP53.
• P037 CLL-1: Targets various genes and regions associated with B-cell chronic lymphocytic leukemia (B-CLL).
• P038 CLL-2: Targets various genes and regions associated with B-cell chronic lymphocytic leukemia (B-CLL).
• P040 CLL: Targets various genes and regions associated with B-cell chronic lymphocytic leukemia (B-CLL).

Related MLPA probemixes that can be used for confirmation

The probemixes below can be used for confirmation because most of the target probes have different ligation sites than those in D006 Multiple Myeloma. Some probes may still have identical ligation sites; contact us for more information if required.

• P047 RB1: Contains probes for each exon of RB1, except exon 15.
• P041 ATM-1: Contains probes for each exon of ATM together with P042 ATM-2.
• P042 ATM-2: Contains probes for each exon of ATM together with P041 ATM-1.
• P088 Oligodendroglioma 1p-19q: Contains probes for 1p, 1q, 19p, and 19q.
• P380 Wilms' tumour: Contains probes for 1p, 1q, 16p, 16q, and TP53.
• P480 WHS & Achondroplasia: Contains probes for 4p16.3, including NSD2 and FGFR3.
• P244 AIP-MEN1-CDKN1B: Contains 6 probes for 12p, including CDKN1B.
• P202 IKZF1-ERG: Contains probes for each exon of IKZF1, 14q32.33, and more.
• P335 ALL-IKZF1: Contains probes for IKZF1, ETV6, RB1, and more.
• P037 CLL-1: Contains probes for 2p, 6q, 8p, 8q (MYC), 11q (ATM), chr. 12, 13q14 (RB1, DLEU2), TP53, and 17p (TP53).
• P038 CLL-2: Contains probes for 11q (ATM), chr. 12, 13q14 (RB1, DLEU1/2), TP53, 14q32.33, 17p (TP53), and chr. 19.
• P040 CLL: Contains probes for 11q (ATM), chr. 12, 13q14 (RB1, DLEU1/2), TP53, and 17p (TP53).
• P258 SMARCB1: Contains probes for each exon of SMARCB1, and flanking regions on 22q.
• P414 MDS: Contains probes for chr. 3, 5q, 7q, 8q, 11q, 12p (ETV6), chr. 17 (TP53), chr. 19, 20q, and chr. Y.
• P323 CDK4-HMGA2-MDM2: Contains probes for 12p and 12q.
• P051 Parkinson mix 1: Contains probes for each exon of PRKN together with P052 Parkinson mix 2.
• P052 Parkinson mix 2: Contains probes for each exon of PRKN together with P051 Parkinson mix 1.
• P064 Microdeletion Syndromes-1B: Contains 9 probes for NSD2, and more.
• P377 Hematologic Malignancies: Contains probes for IKZF1, MYC, ATM, ETV6, 13q14, TP53, and more.
• P078 Breast tumour: Contains probes for MYC, CCND1, and more.
• P451 Chromosome 16: Contains probes for 16p and 16q.
• P343 Autism-1: Contains 26 probes for 15q.
• P298 BRAF-HRAS-KRAS-NRAS: Contains a mutation-specific probe for BRAF p.V600E, and more.
• P301 Medulloblastoma mix 1: Contains probes for chr. 6, 14q, chr. 16, and chr. 17.
• P302 Medulloblastoma mix 2: Contains probes for chr. 2, chr. 3, chr. 7, and chr. 9.
• P303 Medulloblastoma mix 3: Contains probes for chr. 1, 4q, 5q, chr. 8, chr. 10, and chr. 20.

Note that the reference probes included in most of these probemixes have not been optimised for MM samples. If the sample derived from MM harbours multiple copy number alterations on the genomic locations of the reference probes, the normalisation can be compromised and reliable result interpretation will not be possible with this probemix.

Related MLPA probemixes that cannot be used for confirmation

The probemixes below cannot be used for confirmation because most of the target probes have identical ligation sites as those in D006 Multiple Myeloma.

• P425 Multiple Myeloma: Contains probes for 1p, 1q, 5q31, chr. 9, 12p13, 13q14-q22, TRAF3, chr. 15, CYLD, WWOX, TP53.
• P056 TP53: Contains probes for each exon of TP53.
• P105 Glioma: Contains nine probes for TP53, and more.

D006 Multiple Myeloma

• Product flyer
• Data demo set

General digitalMLPA resources

• digitalMLPA technique overview
• Coffalyser digitalMLPA overview
• Explore available digitalMLPA panels

digitalMLPA education

• Getting started with digitalMLPA
• On-demand webinar: digitalMLPA: Where MLPA Meets NGS
• Help centre with other learning resources

Selected publications using D006 Multiple Myeloma

• Croft J et al. (2021). Copy number evolution and its relationship with patient outcome-an analysis of 178 matched presentation-relapse tumor pairs from the Myeloma XI trial. Leukemia. 35:2043-53.
• Kosztolányi S et al. (2018). High-Throughput Copy Number Profiling by Digital Multiplex Ligation-Dependent Probe Amplification in Multiple Myeloma. J Mol Diagn. 20:777-88.
• Menezes K et al. (2020). High-Throughput Molecular Cancer Cell Line Characterization Using Digital Multiplex Ligation-Dependent Probe Amplification for Improved Standardization of in Vitro Research. J Mol Diagn. 22:1179-88.

References

• Al Zaabi EA et al. (2010). Multiplex ligation-dependent probe amplification versus multiprobe fluorescence in situ hybridization to detect genomic aberrations in chronic lymphocytic leukemia: a tertiary center experience. J Mol Diagn. 12:197-203.
• Bernard-Slagter A et al. (2017). Digital Multiplex Ligation-Dependent Probe Amplification for Detection of Key Copy Number Alterations in T- and B-Cell Lymphoblastic Leukemia. J Mol Diagn. 19:659-72.
• Coll-Mulet L et al. (2008). Multiplex ligation-dependent probe amplification for detection of genomic alterations in chronic lymphocytic leukaemia. Br J Haematol. 142:793-801.