SALSA^® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia is a research use only (RUO) assay for the detection of deletions or gains in 73 genes and eight chromosomal regions, as specified in Table 2 of the product description, which are associated with acute lymphoblastic leukemia (ALL).

ALL is the most common childhood cancer comprising multiple genetically heterogeneous subtypes of malignant clonal expansions of immature T- or B-cells. Although current treatment regimens have resulted in 5-year event-free survival rates of >90% in children, disease relapse is associated with a poor outcome (Roberts and Mullighan 2015). The genetic alterations in different subtypes of ALL are well characterised and include gross chromosomal aberrations such as hyper-/hypodiploidy, but also fusion genes, gene deletions, gains and amplifications. SALSA^® digitalMLPA™ Probemix D007 Acute Lymphoblastic Leukemia is designed to detect these key genetic copy number alterations in childhood ALL.

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 D007 Acute Lymphoblastic Leukemia). 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 D007 Acute Lymphoblastic Leukemia version B1. 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 D007 Acute Lymphoblastic Leukemia 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.

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 D007 Acute Lymphoblastic Leukemia. Some probes may still have identical ligation sites; contact us for more information if required.

• SALSA MLPA Probemix P047 RB1: Contains probes for each exon of RB1, except for exon 15.
• SALSA MLPA Probemix P081 NF1 mix 1: Contains probes for each exon of NF1 together with P082 NF1 mix 2.
• SALSA MLPA Probemix P082 NF1 mix 2: Contains probes for each exon of NF1 together with P081 NF1 mix 1.
• SALSA MLPA Probemix P175 Tumour Gain: Contains probes for exons 1 and 12 of ABL1.
• SALSA MLPA Probemix P202 IKZF1-ERG: Contains probes for each exon of IKZF1 and ERG.
• SALSA MLPA Probemix P298 BRAF-HRAS-KRAS-NRAS: Contains nine probes targeting KRAS.
• SALSA MLPA Probemix P323 CDK4-HMGA2-MDM2: Contains probes on chromosomal arms 12p and 12q.
• SALSA MLPA Probemix P327 iAMP21-ERG: Contains probes for the iAMP21 region, including probes for RUNX1 and for each exon of ERG.
• SALSA MLPA Probemix P329 CRLF2-CSF2RA-IL3RA: Contains probes for SHOX, CRLF2, CSF2RA, IL3RA and P2RY8.
• SALSA MLPA Probemix P335 ALL-IKZF1: Contains probes for EBF1, IKZF1, PAX5, ETV6, BTG1, RB1 and the PAR1 region.
• SALSA MLPA Probemix P370 BRAF-IDH1-IDH2: Contains several probes for MYB.
• SALSA MLPA Probemix P377 Hematologic Malignancies: Contains several probes for IKZF1, RUNX1, RB1 and flanking probes DLEU1 and DLEU2.
• SALSA MLPA Probemix P383 T-ALL: Contains probes for TAL1, STIL, LEF1, CASP8AP2, MYB, EZH2, MLLT3, ABL1, NUP214, LMO1/2, RAG2/CD44/SLC1A2, NF1, SUZ12, PTPN2 and PHF6.
• SALSA MLPA Probemix P414 MDS: Contains seven probes for 5q31.2-q33.3 (including additional probes for EGR1 and SPARC).
• SALSA MLPA Probemix P419 CDKN2A/2B-CDK4: Contains multiple probes for each exon of CDKN2A and CDKN2B.
• SALSA MLPA Probemix P437 Familial MDS-AML: Contains one probe for each exon of RUNX1.
• SALSA MLPA Probemix P474 CD274-PDCD1LG2-JAK2: Contains several probes for JAK2.
• SALSA MLPA Probemix ME024 9p21 CDKN2A/2B region: Contains probes for each exon of CDKN2A, CDKN2B and MTAP.

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 D007 Acute Lymphoblastic Leukemia.

• SALSA MLPA Probemix P056 TP53: Contains probes for each exon of TP53.
• SALSA MLPA Probemix P105 Glioma: Contains one probe for each exon of CDKN2A and PTEN. Also contains 9 probes for TP53.
• SALSA MLPA Probemix P225 PTEN: Contains at least two probes for each exon of PTEN.

D007 Acute Lymphoblastic Leukemia

• 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 D007 Acute Lymphoblastic Leukemia

• Antic Ž et al. (2022). Clonal dynamics in pediatric B-cell precursor acute lymphoblastic leukemia with very early relapse. Pediatr Blood Cancer. 69:e29361.
• Bedics G et al. (2023). PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia. Br J Cancer. 129:455-65.
• Benard-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.
• Blunck CB et al. (2024). Characterisation of cells markers associated with IKZF1plus in BCP-ALL. Transl Oncol. 50:102127.
• Kiss R et al. (2020). Comprehensive profiling of disease-relevant copy number aberrations for advanced clinical diagnostics of pediatric acute lymphoblastic leukemia. Mod Pathol. 33:812-24.
• Lopes BA et al. (2023). The recombinome of IKZF1 deletions in B-cell precursor ALL. Leukemia. 37:1727-31.
• Maciel ALT et al. (2022). IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia. Transl Oncol. 15:101291.
• Palmi C et al. (2023). Definition and Prognostic Value of Ph-like and IKZF1plus Status in Children With Down Syndrome and B-cell Precursor Acute Lymphoblastic Leukemia. Hemasphere. 7:e892.
• Thakral D et al. (2019). Rapid Identification of Key Copy Number Alterations in B- and T-Cell Acute Lymphoblastic Leukemia by Digital Multiplex Ligation-Dependent Probe Amplification. Front Oncol. 9:871.
• Zur Stadt U et al. (2019). Characterization of novel, recurrent genomic rearrangements as sensitive MRD targets in childhood B-cell precursor ALL. Blood Cancer J. 9:96.

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.
• Roberts KG et al. (2015). Genomics in acute lymphoblastic leukaemia: insights and treatment implications. Nat Rev Clin Oncol. 12:344-57.