The SALSA MLPA Probemix P335 ALL-IKZF1 is an in vitro diagnostic (IVD) or a research use only (RUO) semi-quantitative 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/B, 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.

Note that the clinical relevance of some genes in the P335 ALL-IKZF1 probemix is not yet fully established. Therefore, the CE mark for diagnostic use only applies to the IKZF1 gene, and all other genes in the probemix are meant to be used in a research setting only.

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).

Several other (partial) gene deletions and duplications, such as in PAX5, ETV6, RB1, BTG1, EBF1 and CDKN2A/2B, have also been described in ALL patients. Prognostic profiles combining these aberrations, such as the IKZF1^plus profile (IKZF1 deletions that co-occur with deletions in CDKN2A, CDKN2B, PAX or PAR1 in the absence of ERG deletion; Stanulla et al. 2018), have been described in recent years. See Table 2 for more information on all genes and regions covered in this probemix.

SALSA MLPA Probemix P335 ALL-IKZF1 is CE-marked for in vitro diagnostic (IVD) use. 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

• SALSA digitalMLPA Probemix 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.
• SALSA MLPA Probemix 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.
• SALSA MLPA Probemix P018 SHOX: Contains probes for the SHOX gene and Xp22 regions.
• SALSA MLPA Probemix P047 RB1: Contains probes for 26 out of 27 exons of the RB1 gene.
• SALSA MLPA Probemix 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.
• SALSA MLPA Probemix P327 iAMP21-ERG: Contains probes for RUNX1, ERG genes and iAMP21 detection in ALL.
• SALSA MLPA Probemix P329 CRLF2-CSF2RA-IL3RA: Contains probes for CRLF2, CSF2RA, IL3RA and SHOX genes, involved in B-ALL.
• SALSA MLPA Probemix P377 Hematologic Malignancies: Contains probes for the most common copy number alterations in ALL, AML, CLL, CML, MDS and various lymphomas.
• SALSA MLPA Probemix 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.
• SALSA MLPA Probemix 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.
• 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.
• Malard F et al. (2020). Acute lymphoblastic leukaemia. Lancet. 395:1146-62.
• 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.
• 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.
• 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.