The SALSA MLPA Probemix P335 ALL-IKZF1 is an in vitro diagnostic (IVD) or a research use only (RUO) semi-quantitative manual assay for the detection of deletions of the IKZF1 gene for stratification of patients with acute lymphoblastic leukemia (ALL) into prognostic subgroups. The SALSA MLPA Probemix P335 ALL-IKZF1 is a RUO assay² for the detection of deletions or duplications in B-cell differentiation and cell cycle control genes (EBF1, CDKN2A/2B, PAX5, ETV6, BTG1 and RB1) and in the PAR1 region. This assay is for use on genomic DNA isolated from human peripheral whole blood and bone marrow specimens.

Certain probes targeting additional genes included in P335 ALL-IKZF1 may only be used in a research setting. The following table summarises which probes are for IVD, and which are exclusively restricted to RUO use.

For the full intended purpose, see the product description.

The overall incidence rate of ALL amounts to 1.6 in 100,000 per year (Malard and Mohty 2020). The peak incidence lies in childhood at an age of less than 5 years; thereafter, the incidence rate declines continually until the age of 50 years. After that, incidence slightly rises a second time. In patients over 50 years it rises a second time and reaches another peak at the age of over 80 years). There is a slight predominance of males (1.2:1). B-cell ALL accounts for 75% of all cases of ALL and T-cell ALL accounts for the remaining 25% of cases (https://www.lls.org/leukemia/acute-lymphoblastic-leukemia/diagnosis/all-subtypes).

Partial or complete deletions of the IKZF1 (IKAROS family zinc finger 1) gene are frequently detected in ALL cases (Mullighan et al. 2008), especially in those patients who also carry the BCR-ABL1 gene fusion (Philadelphia chromosome). IKZF1 deletions can be identified in approximately 70% of the children with Philadelphia chromosome-positive (Ph+) ALL (2-4% of all paediatric ALL cases), in 10-15% of Philadelphia chromosome-negative (Ph−) paediatric ALL, and in 40% of adult B-cell precursor ALL cases (Bernt and Hunger 2014; Lejman et al. 2022; van der Sligte et al. 2015). Deletion of IKZF1 is associated with a poor prognosis in B-ALL patients (Mullighan et al. 2009a) and a higher chance of relapse (Kuiper et al. 2010).

IKZF1 gene, 7p12.2

Deletions or mutations of IKZF1 are significantly associated with an increased risk of relapse and adverse events in ALL patients (Mullighan et al. 2009a; Martinelli et al. 2009).

IKZF1^plus profile

Deletions of IKZF1 in combination with deletions of CDKN2A, CDKN2B, PAX5 and the PAR1 region, and in the absence of ERG deletions, comprise a prognostic profile (IKZF1^plus profile) that is associated with a very poor prognosis (Stanulla et al. 2018). Please note that this probemix does not cover all genes and regions included in the IKZF1^plus profile.

EBF1 gene, 5q33.3

EBF1 deletions are suggested to be an important factor in the relapse of ALL, as 25% of relapsed ALL cases have deletions in the EBF1 locus (Yang et al. 2008). Deletions of EBF1 are suggested to be associated with a higher risk of relapse (Olsson et al. 2014).

CDKN2A/2B genes, 9p21.3

Deletions of CDKN2A are present in 15-35% of childhood B-ALL and in 30-45% of adult B-ALL cases (Gonzáles-Gil et at. 2021). CDKN2A/2B deletions are more frequently found in high-risk patients of all ages, and the majority of studies on relapse ALL suggests that (homozygous) CDKN2A/2B deletions are more frequent at relapse than at diagnosis. Deletions of CDKN2A/2B are also part of the IKZF1^plus profile (see section above).

PAX5 gene, 9p13.2

PAX5 deletions are present in 30% of B-progenitor ALL cases and in 50% of Ph+ ALL (Lejman et al. 2022; Li et al. 2021). Moreover, intragenic PAX5 amplifications were suggested to be present in a new subgroup of B-cell precursor ALL (~1%) and in 3% of B-other ALL cases, which is associated with poor outcome (Schwab et al. 2017). Deletions of PAX5 are also part of the IKZF1^plus profile (see section above).

ETV6 gene, 12p13.2

ETV6 deletions are frequent in B-ALL (51%), and ETV6 is often involved in rearrangements and fusions detected in ALL patients (e.g. in ETV6-RUNX1 fusions) (Schwab et al. 2013). It is suggested that native ETV6 deletions in ETV6-RUNX1+ childhood ALL is associated with better prognosis (Ko et al. 2011). Although microdeletions often occur at the translocation breakpoints, this MLPA probemix will not detect all microdeletions in which ETV6 is involved.

BTG1 gene, 12q21.33

BTG1 deletions appear to be more frequent in high risk ALL cases and are often combined with other deletions (Fang et al. 2018). BTG1 deletions are detected with high frequency in ALL patients with Down syndrome (Lundin et al. 2012). As many deletions of BTG1 extend into the centromeric area of BTG1 into a gene poor region (Waanders et al. 2012), two downstream probes were included for the BTG1-area.

RB1 gene, 13q14.2

RB1 deletions have been reported to be more frequent in high risk ALL as compared to non-selected cases and often co-occur with other CNAs, in particular iAMP21 (Fang et al. 2018; Schwab et al. 2013; Steeghs et al. 2019). Deletions often involve one or more of the last 10 exons of this 27-exon gene (Schwab et al. 2013).

Xp22.33 / Yp11.32 (PAR1) region

This region shows recurrent aberrations in ALL (Harvey et al. 2010). CSF2RA/IL3RA deletions are frequent in ALL (7% of ALL cases; up to 55% in Down syndrome-associated ALL). Focal deletions within the P2RY8 and CRLF2 genes, resulting in a fusion gene, have been suggested to associate with a poor prognosis (Mullighan et al. 2009b). Deletions of the PAR1 region are also part of the IKZF1^plus profile (see section above).

SALSA MLPA Probemix P335 ALL-IKZF1 is CE-marked under the IVDD for in vitro diagnostic (IVD) use in Europe. This assay has also been registered for IVD use in Colombia, Costa Rica and Israel.

This assay is for research use only (RUO) in all other 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 have been tested with the current (C2) version of this product and have been shown to produce useful results.

• Coriell NA01353 (m): Heterozygous deletion affecting the probes for SHOX, CRLF2 and CSF2RA on chromosome Xp (PAR1 region).
• Coriell NA01750 (m): Heterozygous duplication affecting the probes for JAK2, CDKN2A and CDKN2B on chromosome 9p.
• Coriell NA04371 (m): Heterozygous duplication affecting the probes for EBF1 on chromosome 5q and CSF2RA on chromosome Xp (PAR1 region).
• Coriell NA05067 (m): Heterozygous duplication affecting the probes for JAK2, CDKN2A, CDKN2B and PAX5 on chromosome 9p.
• Coriell NA07081 (m): Heterozygous duplication affecting the probes for IKZF1 on chromosome 7p.
• Coriell NA07981 (m): Heterozygous triplication / homozygous duplication affecting the probes for ETV6 on chromosome 12p.
• Coriell NA09403 (f): Heterozygous deletion affecting the probes for SHOX, CRLF2, CSF2RA, IL3RA and P2RY8 on chromosome Xp (PAR1 region).
• Coriell NA10925 (m): Heterozygous deletion affecting the probes for IKZF1 on chromosome 7p. Heterozygous duplication affecting the probes for CRLF2 and CSF2RA on chromosome Xp (PAR1 region).
• Coriell NA12606 (m): Heterozygous duplication affecting the probes for RB1 on chromosome 13q.
• Coriell NA12722 (m): Heterozygous duplication affecting the probes for JAK2, CDKN2A and CDKN2B on chromsome 9p. Some of the reference probes are affected by CNAs.
• Coriell NA14164 (f): Heterozygous deletion affecting the probes for RB1 on chromosome 13q.
• DSMZ ACC-20 (BV-173) (f): Heterozygous deletion affecting the probes for IKZF1 (exons 1-7) on chromosome 7p, CDKN2A (exon 4) on chromosome 9p and probes targeting the PAR1 region on chromosome Xp. Homozygous deletion affecting the probes for CDKN2A (exon 2) and CDKN2B (exon 2) on chromosome 9p. Some of the reference probes are affected by CNAs.

Various related products

• D007 Acute Lymphoblastic Leukemia: DigitalMLPA probemix that contains probes for 52 target genes and three chromosomal regions associated with ALL, including 16 probes for IKZF1.
• ME024 9p21 CDKN2A/2B region: Contains probes for the 9p21 region, including CDKN2A and CDKN2B genes for detection of both copy number and methylation status.
• P018 SHOX: Contains probes for the SHOX gene and Xp22 regions.
• P047 RB1: Contains probes for 26 out of 27 exons of the RB1 gene.
• P202 IKZF1-ERG: Contains two probes for all exons and the regulatory regions of IKZF1 transcript variant 1 (NM_006060.6), one probe for each exon of the ERG gene, and probes for CDKN2A/2B genes and the 14q32.33 region.
• P327 iAMP21-ERG: Contains probes for RUNX1, ERG genes and iAMP21 detection in ALL.
• P329 CRLF2-CSF2RA-IL3RA: Contains probes for CRLF2, CSF2RA, IL3RA and SHOX genes, involved in B-ALL.
• P377 Hematologic Malignancies: Contains probes for the most common copy number alterations in ALL, AML, CLL, CML, MDS and various lymphomas.
• P383 T-ALL: Contains probes for STIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, MLLT3, MTAP, CDKN2A/2B, NUP214-ABL1, PTEN, LMO1/2, NF1, SUZ12, PTPN2 andPHF6 genes, involved in T-ALL.
• P419 CDKN2A/2B-CDK4: Contains probes for CDKN2A, CDKN2B and CDK4 genes.

General MLPA resources

• MLPA technique overview
• Coffalyser.Net overview

MLPA education

• Getting started with MLPA
• Using Coffalyser.Net to analyse MLPA data
• Help centre with other learning resources

Selected publications using P335 ALL-IKZF1

• Ayón-Pérez MF et al. (2019). IKZF1 Gene Deletion in Pediatric Patients Diagnosed with Acute Lymphoblastic Leukemia in Mexico. Cytogenet Genome Res. 158:10-6.
• Crepinsek K et al. (2021). Clinical impacts of copy number variations in B-cell differentiation and cell cycle control genes in pediatric B-cell acute lymphoblastic leukemia: a single centre experience. Radiol Oncol. 56:92-101.
• Fang Q et al. (2021). Gene Deletions and Prognostic Values in B-Linage Acute Lymphoblastic Leukemia. Front Oncol. 11:677034.
• Gupta SK et al. (2018). Molecular genetic profile in BCR-ABL1 negative pediatric B-cell acute lymphoblastic leukemia can further refine outcome prediction in addition to that by end-induction minimal residual disease detection. Leuk Lymphoma. 59:1899-1904.
• Krentz S et al. (2013). Prognostic value of genetic alterations in children with first bone marrow relapse of childhood B-cell precursor acute lymphoblastic leukemia. Leukemia. 27:295-304.
• Titieanu AA et al. (2023). Prognostic Significance of the Number of Copy Numbervariations by Mlpa Technique in Acute Lymphoblastic Leukemia. Documenta Haematologica - Revista Romana de Hematologie. 1:43-9.
• Yamashita Y et al. (2013). IKZF1 and CRLF2 gene alterations correlate with poor prognosis in Japanese BCR-ABL1-negative high-risk B-cell precursor acute lymphoblastic leukemia. Pediatr Blood Cancer. 60:1587-92.
• Yu CH et al. (2020). MLPA and DNA index improve the molecular diagnosis of childhood B-cell acute lymphoblastic leukemia. Sci Rep. 10:11501.

References

• Bernt KM et al. (2014). Current concepts in pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia. Front Oncol. 4:54.
• Fang Q et al. (2018). Prognostic significance of copy number alterations detected by multi-link probe amplification of multiple genes in adult acute lymphoblastic leukemia. Oncol Lett. 15:5359-67.
• González-Gil C et al. (2021). The Yin and Yang-Like Clinical Implications of the CDKN2A/ARF/CDKN2B Gene Cluster in Acute Lymphoblastic Leukemia. Genes (Basel). 12:79.
• Harvey RC et al. (2010). Rearrangement of CRLF2 is associated with mutation of JAK kinases, alteration of IKZF1, Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute lymphoblastic leukemia. Blood. 115:5312-21.
• Ko DH et al. (2011). Native ETV6 deletions accompanied by ETV6-RUNX1 rearrangements are associated with a favourable prognosis in childhood acute lymphoblastic leukaemia: a candidate for prognostic marker. Br J Haematol. 155:530-3.
• Kuiper RP et al. (2010). IKZF1 deletions predict relapse in uniformly treated pediatric precursor B-ALL. Leukemia. 24:1258-64.
• Lejman M et al. (2022). Genetic Biomarkers and Their Clinical Implications in B-Cell Acute Lymphoblastic Leukemia in Children. Int J Mol Sci. 23:2755.
• Li J et al. (2021). Emerging molecular subtypes and therapeutic targets in B-cell precursor acute lymphoblastic leukemia. Front Med. 15:347-71.
• Lundlin C et al. (2012). High frequency of BTG1 deletions in acute lymphoblastic leukemia in children with down syndrome. Genes Chromosomes Cancer. 51:196-206.
• Malard F et al. (2020). Acute lymphoblastic leukaemia. Lancet. 395:1146-62.
• Martinelli G et al. (2009). IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are associated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report. J Clin Oncol. 27:5202-7.
• Mullighan CG et al. (2008). BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature. 453:110-4.
• Mullighan CG et al. (2009a). Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med. 360:470-80.
• Mullighan CG et al. (2009b). Rearrangement of CRLF2 in B-progenitor- and Down syndrome-associated acute lymphoblastic leukemia. Nat Genet. 41:1243-6.
• Olsson L et al. (2014). Deletions of IKZF1 and SPRED1 are associated with poor prognosis in a population-based series of pediatric B-cell precursor acute lymphoblastic leukemia diagnosed between 1992 and 2011. Leukemia. 28:302-10.
• Schwab C et al. (2017). Intragenic amplification of PAX5: a novel subgroup in B-cell precursor acute lymphoblastic leukemia? Blood Adv. 1:1473-7.
• Schwab CJ et al. (2013). Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features. Haematologica. 98:1081-8.
• Stanulla M et al. (2018). IKZF1^plus Defines a New Minimal Residual Disease-Dependent Very-Poor Prognostic Profile in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia. J Clin Oncol. 36:1240-9.
• Steeghs EMP et al. (2019). Copy number alterations in B-cell development genes, drug resistance, and clinical outcome in pediatric B-cell precursor acute lymphoblastic leukemia. Sci Rep. 9:4634.
• Van der Sligte NE et al. (2015). Effect of IKZF1 deletions on signal transduction pathways in Philadelphia chromosome negative pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Exp Hematol Oncol. 4:23.
• Waanders E et al. (2012). The origin and nature of tightly clustered BTG1 deletions in precursor B-cell acute lymphoblastic leukemia support a model of multiclonal evolution. PLoS Genet. 8:e1002533.
• Yang JJ et al. (2008). Genome-wide copy number profiling reveals molecular evolution from diagnosis to relapse in childhood acute lymphoblastic leukemia. Blood. 112:4178-83.