NXtec 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. NXtec 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 NXtec 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 NXtec 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.

NXtec 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.

The commercially available positive samples below can be used with the current (B1) version of this product.

• Coriell NA00501 (m): Heterozygous duplication affecting the probes for LEF1, INPP4B, ZNF827, NR3C2, CYP4V2 and TRIML1 on chromosome 4q.
• Coriell NA00526 (m): Trisomy of chromosome 13 affecting the probes for KATNAL1, ITM2B, RB1, RCBTB2, CDADC1, DLEU2, DLEU1, VPS36, KLHL1, ARHGEF7 and GRK1 on chromosome 13q.
• Coriell NA00782 (m):
  • Heterozygous duplication affecting the probes for TECRL, LEF1, INPP4B and ZNF827 on chromosome 4q.
  • Heterozygous duplication affecting the probes for CSF2RA on chromosome Xp.
• Coriell NA00803 (m): Heterozygous deletion affecting the probes for MEF2D and PBX1 on chromosome 1q.
• Coriell NA00981 (f): Heterozygous duplication affecting the probes for RSPO4, TGM6, TRMT6, PLCB4, RIN2 and APMAP on chromosome 20p.
• Coriell NA01221 (m): Heterozygous duplication affecting the probes for COL19A1, RIMS1, CASP8AP2, TBC1D32 and LAMA2 on chromosome 6q.
• Coriell NA01359 (m): Heterozygous duplication affecting the probes for CLUL1, LPIN2, MYOM1, GNAL, SPIRE1, PTPN2, LDLRAD4, FAM210A, RBBP8, NPC1, LOXHD1, LIPG, CTDP1 and TXNL4A on chromosome 18.
• Coriell NA02030 (m): Trisomy of chromosome 8 affecting the probes for FBXO25, CLN8, GFRA2, CDCA2, RBPMS, GSR, SNTG1, TOX, CHD7, RMDN1, CPNE3, TSNARE1 and SLC39A4 on chromosome 8.
• Coriell NA02587 (m):
  • Heterozygous deletion affecting the probes for MYO18A, NF1, SUZ12 and PSMD11 on chromosome 17q.
  • Heterozygous duplication affecting the probes for SHOX, SHOX-AREA, CRLF2, CSF2RA, IL3RA, P2RY8, AKAP17A, ASMT, ZBED1 and CD99 on chromosome Xp.
  • Gain affecting the probes for SRY, PCDH11Y, TBL1Y, USP9Y, KDM5D and RPS4Y2 on chromosome Y.
• Coriell NA02718 (f):
  • Heterozygous deletion affecting the probes for ITM2B, RB1, RCBTB2, CDADC1, DLEU2, DLEU1 and VPS36 on chromosome 13q.
  • Heterozygous deletion affecting the probes for ACOT9 and DMD on chromosome Xp.
• Coriell NA02819 (f):
  • Heterozygous duplication affecting the probes for DOCK8, KANK1, JAK2, GLDC, FREM1, BNC2, MLLT3, MTAP, CDKN2A and CDKN2B on chromosome 9p.
  • Heterozygous deletion affecting the probes for GALNT9 and PGAM5 on chromosome 12q.
• Coriell NA03184 (m): Trisomy of chromosome 15 affecting the probes for ATP10A, OCA2, SPRED1, VPS13C, USP3, BLM, CHSY1 and TM2D3 on chromosome 15.
• Coriell NA03226 (m): Heterozygous duplication affecting the probes for DOCK8, KANK1, JAK2, GLDC, FREM1, BNC2, MLLT3, MTAP, CDKN2A, CDKN2B PAX5, ZCCHC7, FBXO10 and DCAF10 on chromosome 9p.
• Coriell NA03330 (m): Trisomy of chromosome 13 affecting the probes for CDX2, URAD, FLT3, PAN3, PAN3-AS1, FLT1, KATNAL1, ITM2B, RB1, RCBTB2, CDADC1, DLEU2, DLEU1, VPS36, KLHL1, ARHGEF7 and GRK1 on chromosome 13q.
• Coriell NA03493 (f):
  • Heterozygous deletion affecting the probes for HNRNPH1, SQSTM1, MAPK9 and FLT4 on chromosome 5q.
  • Heterozygous duplication affecting the probes for SERPINB6 and ECI2 on chromosome 6p.
• Coriell NA03563 (m):
  • Heterozygous deletion affecting the probes for CD200, BTLA, XRN1, SLC9A9, TBL1XR1, LINC00501, OPA1 and ACAP2 on chromosome 3q.
  • Heterozygous deletion affecting the probes for DOCK8 and KANK1 on chromosome 9p.
• Coriell NA04371 (m):
  • Heterozygous duplication affecting the probes for EBF1, COL23A1, SQSTM1, MAPK9 and FLT4 on chromosome 5q.
  • Heterozygous duplication affecting the probes for CSF2RA on chromosome Xp.
• Coriell NA04626 (f): Trisomy of chromosome X affecting the probes for SHOX, SHOX-AREA, CRLF2, CSF2RA, IL3RA, P2RY8, AKAP17A, ASMT, ZBED1, CD99, GYG2, ANOS1, ACOT9, DMD, FGD1, ARHGEF9, ZDHHC15, NXF3, PHF6, CLIC2 and TMLHE on chromosome X.
• Coriell NG04823 (m): Trisomy of chromosome 21 affecting the probes for RBM11, HSPA13, SAMSN1, MIR99A, BTG3, TMPRSS15, NCAM2, MIR155, APP, CYYR1, ADAMTS5, BACH1, TIAM1, OLIG2, ITSN1, KCNE2, RUNX1, SIM2, HLCS, DYRK1A, KCNJ6, ERG, ETS2, PSMG1, TMPRSS2, RIPK4, TFF1, PDE9A, TRAPPC10, TSPEAR, ITGB2, SLC19A1, COL6A2 and PRMT2 on chromosome 21q.
• Coriell NA06226 (m): Heterozygous duplication affecting the probes for DECR2, IFT140, CREBBP, TXNDC11 and CPPED1 on chromosome 16p.
• Coriell NA06803 (m): Heterozygous deletion affecting the probes for LMO2, CD44, SLC1A2 and RAG2 chromosome 11p.
• Coriell NA07081 (m): Heterozygous duplication affecting the probes for SDK1, RADIL, RAPGEF5, STK31, ADCY1, ABCA13, IKZF1, LANCL2 and SUMF2 on chromosome 7p.
• Coriell NA07106 (m): Trisomy of chromosome 22 affecting the probes for GAB4, CECR2, TOP3B, PRAMENP, IGLV8-61, IGLV4-60, VPREB1, BMS1P20, IGLV7-43, IGLL1, ZMAT5, SFI1, TRMU and BRD1.
• Coriell NA07412 (m): Heterozygous deletion affecting the probes for EPHA1, EZH2, RBM33 and DYNC2I1 on chromosome 7q.
• Coriell NA07994 (m):
  • Heterozygous duplication affecting the probes for MYB, SMOC2 and ERMARD on chromosome 6q.
  • Heterozygous deletion affecting the probes for INPP5A and KNDC1 on chromosome 10q.
• Coriell NA08146 (f):
  • Heterozygous deletion affecting the probes for RAI1 and ULK2 on chromosome 17p.
  • Heterozygous deletion affecting the probes for VPREB1 on chromosome 22q.
  • Heterozygous duplication affecting the probes for CRLF2 and CSF2RA on chromosome Xp. One of the SHOX-AREA probes is also affected.
• Coriell NA08778 (m): Heterozygous deletion affecting the probes for EPHA6, CPOX, CD200 and BTLA on chromosome 3q.
• Coriell NA09216 (m):
  • Heterozygous deletion affecting the probes for DNMT3A on chromosome 2p.
  • Gain affecting the probe for ASMT on chromosome Xp.
• Coriell NA10401 (f): Trisomy of chromosome 2 affecting the probes for TMEM18, COLEC11, SPAST, VIT, FER1L5, CNNM3, NPAS2, SCN1A, ABCB11, IKZF2, CAPN10 and KIF1A on chromosome 2.
• Coriell NA10925 (m):
  • Heterozygous deletion affecting the probes for ADCY1, ABCA13 and IKZF1 on chromosome 7p.
  • Heterozygous duplication affecting the probes for CRLF2 and CSF2RA exon 1-11 on chromosome Xp.
• Coriell NA10989 (m):
  • Heterozygous deletion affecting the probes for DOCK8, KANK1, JAK2 and GLDC on chromosome 9p.
  • Heterozygous deletion affecting the probes for IGHM on chromosome 14q.
• Coriell NA12722 (m):
  • Trisomy 9 and a partial heterozygous deletion of chromosome 9. The probes for DOCK8, KANK1, JAK2, GLDC, FREM1, BNC2, MLLT3, MTAP, CDKN2A, CDKN2B, PGM5, TRPM3, ANKS6, ALDOB, ABL1, NUP214, NOTCH1, GRIN1 and EHMT1 appear to be heterozygously duplicated. The probes for PAX5, ZCCHC7, FBXO10 and DCAF10 appear not to be affected by copy number changes.
  • Trisomy 18 affecting the probes for CLUL1, LPIN2, MYOM1, GNAL, SPIRE1, PTPN2, LDLRAD4, FAM210A, RBBP8, NPC1, LOXHD1, LIPG, CTDP1 and TXNL4A.
• Coriell NA13031 (m):
  • Deletion affecting the probes for IGHM on chromosome 14q.
  • Heterozygous deletion affecting the probes for HLF and ANKFN1 on chromosome 17q.
  • Heterozygous duplication affecting the probe for KCNE2 on chromosome 21q.
• Coriell NA13284 (m):
  • Heterozygous duplication affecting the probe for DECR2 on chromosome 16p.
  • Heterozygous deletion affecting the probes for TRMU and BRD1 on chromosome 22q.
• Coriell NA13410 (m):
  • Heterozygous duplication affecting the probes for DYNC1H1, COA8 and IGHM on chromosome 14q.
  • Heterozygous deletion affecting the probes for UCKL1 on chromosome 20q.
  • Homozygous duplication affecting the probe for ASMT on chromosome Xp.
• Coriell NA13685 (f):
  • Heterozygous deletion affecting the probe for DYNC2I1 on chromosome 7q.
  • Heterozygous duplication affecting the probes for ABL1, NUP214, NOTCH1, GRIN1 and EHMT1 on chromosome 9q.
  • Heterozygous deletion affecting the probes for IGHM on chromosome 14q.
• Coriell NA14523 (f):
  • Heterozygous duplication affecting the probes for IRX4, NSUN2, DNAH5, DROSHA and NPR3 on chromosome 5p.
  • Heterozygous deletion affecting the probes for SHOX, SHOX-AREA, CRLF2, CSF2RA, IL3RA, P2RY8, AKAP17A, ASMT, ZBED1, CD99, GYG2, ANOS1, ACOT9, DMD and FGD1 on chromosome Xp.
• Coriell NA16362 (m):
  • Heterozygous duplication affecting the probes for GAB4, CECR2, TRMU and BRD1 on chromosome 22q.
  • Heterozygous deletion affecting the probes for TOP3B, PRAMENP, IGLV8-61 and IGLV4-60 on chromosome 22q.
• Coriell NA17941 (f):
  • Heterozygous duplication affecting the probes for BCL9, GJA5, RPRD2, SMCP, NUP210L, LMNA, MEF2D, PBX1, KCNT2, DENND1B, ADSS2 and DESI2 on chromosome 1q.
  • Heterozygous deletion affecting the probes for IGHM on chromosome 14q.
  • Heterozygous deletion affecting the probes for NXF3, PHF6, CLIC2 and TMLHE on chromosome Xq.
• Coriell NA20027 (f):
  • Heterozygous deletion affecting the probes for IGHM on chromosome 14q.
  • Heterozygous deletion affecting the probes for SHOX, SHOX-AREA, CRLF2, CSF2RA, IL3RA, P2RY8, AKAP17A, ASMT, ZBED1, CD99, GYG2, ANOS1, ACOT9, DMD, FGD1, ARHGEF9, ZDHHC15, NXF3, PHF6, CLIC2 and TMLHE on chromosome X.
• Coriell NA20125 (m):
  • Heterozygous duplication affecting the probes for PTEN, ADD3, INPP5A and KNDC1 on chromosome 10q.
  • Heterozygous deletion affecting the probes for IGHM on chromosome 14q.
• Coriell NA50322 (f):
  • Heterozygous deletion affecting the probes for IGHM on chromosome 14q.
  • Heterozygous deletion affecting the probes for CLUL1, LPIN2, MYOM1, GNAL, SPIRE1, PTPN2, LDLRAD4 and FAM210A on chromosome 18p.
  • Heterozygous deletion affecting the probe for ADAMTS5 on chromosome 21q.

Key related products

• P335 ALL-IKZF1: Targets IKZF1, CDKB2A/B, PAX5, ETV6, BTG1, RB1, and the PAR1 region, associated with acute lymphoblastic leukemia (ALL).
• P202 IKZF1-ERG: Targets IKZF1, ERG, CDKN2A/B, and 14q32.33, associated with acute lymphoblastic leukemia (ALL).
• P327 iAMP21-ERG: Targets RUNX1, ERG, and iAMP21 detection, associated with acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML).
• P329 CRLF2-CSF2RA-IL3RA: Targets CRLF2, CSF2RA, IL3RA, and SHOX, associated with B-cell acute lymphoblastic leukemia (B-ALL).
• P377 Hematologic Malignancies: Targets the most common copy number alterations associated with acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML), chronic lymphoid leukaemia (CLL), chronic myeloid leukaemia (CML), myelodysplastic syndrome (MDS) and various lymphomas.
• P383 T-ALL: Targets various regions associated with T-lineage acute lymphoblastic leukemia (T-ALL).
• 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 D007 Acute Lymphoblastic Leukemia. 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.
• P081 NF1 mix 1: Contains probes for each exon of NF1 together with P082 NF1 mix 2.
• P082 NF1 mix 2: Contains probes for each exon of NF1 together with P081 NF1 mix 1.
• P175 Tumour Gain: Contains probes for exons 1 and 12 of ABL1.
• P202 IKZF1-ERG: Contains probes for each exon of IKZF1 and ERG.
• P298 BRAF-HRAS-KRAS-NRAS: Contains 9 probes targeting KRAS.
• P323 CDK4-HMGA2-MDM2: Contains probes for 12p and 12q.
• P327 iAMP21-ERG: Contains probes for the iAMP21 region, including probes for RUNX1 and for each exon of ERG.
• P329 CRLF2-CSF2RA-IL3RA: Contains probes for SHOX, CRLF2, CSF2RA, IL3RA and P2RY8.
• P335 ALL-IKZF1: Contains probes for EBF1, IKZF1, PAX5, ETV6, BTG1, RB1 and the PAR1 region.
• P370 BRAF-IDH1-IDH2: Contains several probes for MYB.
• P377 Hematologic Malignancies: Contains several probes for IKZF1, RUNX1, RB1 and flanking probes for DLEU1 and DLEU2.
• 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.
• P414 MDS: Contains 7 probes for 5q31.2-q33.3, including additional probes for EGR1 and SPARC.
• P419 CDKN2A/2B-CDK4: Contains multiple probes for each exon of CDKN2A and CDKN2B.
• P437 Familial MDS-AML: Contains probes for each exon of RUNX1.
• 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.

• P056 TP53: Contains probes for each exon of TP53.
• P105 Glioma: Contains probes for each exon of CDKN2A and PTEN, and 9 probes for TP53.
• P225 PTEN: Contains at least 2 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.