Multiprobe AML/MDS Panel
The Chromoprobe Multiprobe® AML/MDS panel has been designed to detect up to eight different FISH probes on a single slide in a single hybridisation experiment. It can be used to determine genotype in leukaemia patients and aid in prognosis and disease management.
The panel has been designed to aid in the detection of chromosomal aberrations which occur in 30-50% and 60% of MDS and AML cases respectively, thereby providing critical information for diagnosis and prognosis. The product can also be used to monitor the course of the disease by detecting the remission, relapse, disease progression, BMT success, secondary leukaemia and Minimal Residual Disease (MRD).
The orientation of the probes on the panel is illustrated above. More detailed information on the probes can be found below.
- Area of Interest*
- AML, MDS
Deletion of a region of chromosome 5q, which includes the EGR1 gene, is one of the most common rearrangements in AML and MDS1,2.
The deletions are large, with breakpoints occurring between bands 5q11 and 5q35, and are usually interstitial. Two regions, 5q12-14 and 5q31-33, are hot spots for breakpoints but the common deleted region (CDR) in AML and aggressive MDS cases is within 5q31.12,3. Early Growth Response 1 (EGR1) maps to this band3 and loss of this gene may cause tumourigenesis in at least two ways: Firstly, EGR1 directly controls the expression of fibronectin (FN1) through pathways that involve TGFB1 and plasminogen activator inhibitor-1 (SERPINE1 or PAI1). EGR1 dependent expression of TGFB1 inhibits human cancer cell growth in model cells4. In addition, EGR1 is required for p53-dependent apoptosis through the mediation of Retinoblastoma (RB1) protein5.
1. Boultwood J et al., Blood 2002;99(12):4638-41
2. Charrin C, Atlas Genet Cytogenet Oncol Haematol 1998;2(3):302-6
3. OMIM :http://omim.org/entry/153550
4. Liu C et al., Proc Natl Acad Sci USA 1996;93:11831-6
5. Das et al., J. Biol. Chem. 2001 276: 3279-3286
PML/RARα (RARA) Translocation, Dual Fusion
The fusion gene PML/RARA is created by the t(15;17)(q24.1;q21.2) translocation, found in 98% of AML M3 acute hypergranular promyelocytic leukaemia and 9% of AML overall1,2,3.
Breakpoints in the PML gene vary between intron 3 and exon 7a, which is in contrast to the RARA breakpoint, which remains constant in intron 2. Variant translocations include partner breakpoints in 11q23, 5q32 and 11q13. The presence of three-way translocations indicate that the critical event occurs on the der(15), which always receives the distal end of chromosome 17.
The PML protein is a transcription factor and RARA encodes a nuclear receptor. The fusion protein generated, PML-RARA, is a chimaeric transcription factor that operates as a dominant negative form of RARA and results in cells that are blocked at the promyelocytic stage of differentiation and then proliferate. Gain of function mutation allows repression of multiple genes and recruitment of DNA methyltransferases to promoters, allowing prolonged suppression. PML-RARA also activates components of the WNT signalling pathway, promoting stem cell renewal.
Immediate treatment of PML/RARA positive patients is critical as intravascular coagulation causes early death in 10-40% of cases2. Terminal differentiation is induced by the use of all-trans-retinoic-acid (ATRA), which reactivates the RARA gene and degrades the PML-RARA fusion product and 80-90% of treated patients achieve complete remission1,2,4. Despite the efficacy of ATRA, the death rate remains around 15-20%, but 70% of patients will achieve 3 year survival2. Additional abnormalities found with PML/RARA translocations include trisomy 8, seen in one third of cases, del(7q) and del(9q)2.
1. Licht, Sternberg, The Molecular Pathway of AML. ASH Education Book 2005;137-42
2. Huret and Chomienne. t(15;17)(q24;q21). Atlas Genet Cytogenet Oncol Haematol 1998;2(3):329-34
3. Heim and Mitelman, Willey-Liss, Inc. 1995 4. Greaves, BMJ 2002;324(7332):283-7
P53 (TP53) Deletion
Although previously difficult to detect, the advent of FISH analysis of interphase cells from patients with B-CLL showed that around 17% of patients with the disease have deletions of the P53 (TP53) gene1. As with ATM, deletions of P53 have important therapeutic implications for patients with B-CLL2.
Knowledge of the P53 deletion status in the patient should mediate the choice of therapy3. P53 is a tumour suppressor gene and its protein product is responsible for the death of cells that contain damaged DNA. This is thought to be brought about by phosphorylation of P53 and the subsequent prevention of its repression by MDM2 (Mouse Double Minute 2 Homolog). This phosphorylation is mediated by ATM. In the absence of P53 activity, cells that cannot be repaired by ATM will continue to proliferate in their damaged state. Patients deleted for P53 may be rendered resistant to alkylating chemotherapeutic agents3 and purine analogues4 as these are designed to damage DNA in the cells that P53 would have destroyed. In the absence of P53, therefore, patients treated with these agents will harbour a proliferating population of damaged cells.
1. Döhner et al., J Mol Med 1999;77:266-81
2. Foá et al., Haematol 2013; 98(5):675-685
3. Sturm et al., Cell Death Differ. 2003 Apr;10(4):477-84
4. Döhner et al., Blood. 1995 Mar 15;85(6):1580-9
AML1/ETO (RUNX1/RUNX1T1) Translocation, Dual Fusion
AML1 (RUNX1 - Runt related Transcription Factor 1) is fused with ETO (RUNX1T1) in the t(8;21)(q21;q22) translocation, found most commonly in AML M2 patients and less frequently in subgroups M1 and M41.
Overall, 7% of AML cases demonstrate the abnormality, the majority of which are de novo1. Additional abnormalities occur in around 80% of cases - these may be loss of a sex chromosome, del(9q), trisomy 8 or monosomy 71. Three-way variants of this rearrangement, along with the t(3;21) (AML1/EVI1, also known as RUNX1/MECOM) show that juxtaposition of AML1 to the derivative chromosome is consistent and therefore the critical part of the rearrangement2,3. AML1 is the most common target for translocations in acute myeloid leukaemia. The breakpoint mainly occurs in the intron between exons 5 and 6 just before the transactivation domain. The fusion proteins created contain the DNA-binding domain of AML1 fused to the transcription factors ETO or EVI1 (on chromosomes 8 and 3 respectively)1. These abnormalities can give rise to tumourigenic growth through a number of different mechanisms.
1. Huret JL . t(8;21)(q22;q22). Atlas Genet Cytogenet Oncol Haematol. September 1997
2. Nucifora G et al., Blood 1995;86(1):1-14
3. Heim and Mitelman, Willey-Liss, Inc. 1995
Rearrangement of the MLL (KMT2A: lysine (K)-specific methyltransferase 2A) gene on chromosome 11q23.3 can be detected in the leukaemic cells of approximately 85% of infants with B-ALL1,2,3. They can also be found in 3% of de novo and 10% of therapy related AML cases4. Translocations involving the MLL gene are generally associated with increased risk of treatment failure5.
In infant ALL, the most frequent of these translocations is the t(4;11)(q21;q23.3) translocation involving MLL and the AFF1 (AF4) gene on chromosome 46,7. A poor outcome for infants with ALL is strongly associated with the presence of this rearrangement in particular6. The discovery that a single YAC spanned breakpoints in four of the more common translocations led to the naming of the candidate gene MLL (Myeloid/Lymphoid or Mixed Lineage Leukaemia). The gene has homology with a drosophila gene ('trithorax'), which is highly conserved in humans and gives rise to a protein that can be folded to give six zinc finger domains and is a developmental regulator. The zinc finger domains are translocated to the AFF1, MLLT3 and MLLT1 genes on the partner chromosomes in the aforementioned t(4;11), t(9;11)(p22;q23.3) and t(11;19)(q23.3;p13.3) translocations, respectively. Each of the genes involved in these translocations have been shown to have high sequence homology. There have been over 85 recurrent translocations involving the MLL gene reported, with 66 partner genes so far identified8.
1. Rubnitz et al., Blood 1994;84(2):570-3
2. Secker-Walker et al., Leukaemia 1998;12(5):840-4
3. Rowley, Annu Rev Genet 1998;32:495-519
4. Grossman et al., Leukemia 28 March 2013; doi10.1038/leu.2013.90
5. Pui and Evans, New Engl J Med 1998;339(9):605-15
6. Felix and Lange, Oncologist 1999;4(3):225-40
7. Heerema et al., Leukemia 1999;13(5):679-86
8. Huret JL. KMT2A (myeloid/lymphoid or mixed lineage leukemia). Atlas Genet Cytogenet Oncol Haematol. October2005
Del (7q) Deletion
Abnormalities of chromosome 7 are very common in myeloid malignancies, such as adult and paediatric MDS and treatment related AML/MDS.
In children, it is often associated with juvenile Chronic Myeloid Leukaemia (jCML)1,2,3,4. There is also a predominance in leukaemias associated with a constitutional predisposition, caused by disorders including neurofibromatosis 1 (NF1), Fanconi Anaemia and possibly Down syndrome, which produces a distinct clinical picture known as Monosomy 7 syndrome. Another rearrangement, -5/del(5q), is found as an additional abnormality in 40-60% of secondary MDS cases. +8 is less frequently seen4.
Studies of myeloid disorders involving -7/del(7q) have found that signaling pathways involving RAS proteins are affected. There are two commonly deleted regions (CDR): one at 7q22, the other at 7q31-342,3,5,6,7. RELN (7q22) encodes a large secreted protein related to extracellular matrix proteins, a family of proteins that contains multiple epidermal growth factor (EGF)-like proteins.
1. Heim and Mittelman, Willey-Liss, Inc. 1995
2. Emerling BM et al., Oncogene 2002;21:4849-54
3. Kratz CP et al., Genomics 2001;77(3):171-80
4. Desangles F, -7/del(7q) in adults. Atlas Genet Cytogenet Oncol Haematol 1999
5. Le Beau MM et al., Blood 1996;88(6):1930-5
6. Fischer K et al., Blood 1997;89(6):2036-41
7. Koike M et al., Leukemia Res 1999;23:307-10
CBFß/MYH11 Translocation, Dual Fusion
The fusion gene CBFβ (CBFB)/MYH11, created by the inversion inv(16)(p13.11q22.1), is found in 20% of AML M4 cases – in particular M4 with marked eosinophilia (M4eo) - and rarely, in M2, M5 and M4 without eosinophilia1.
Overall, abnormalities involving 16q22 are seen in 5-10% of AML1,2,3. Frequently, Central Nervous System (CNS) involvement develops, particularly in relapse. However, the complete remission rate is high and the prognosis is better than most of the AML associated abnormalities3.
The inversion may be missed in poor cytogenetic preparations. FISH probes for 16p13 often show a deletion within 16p13 in addition to the 16p13.11/16q22.1 rearrangement (~20% cases). In these patients, the split signal may be lost. Variant rearrangements are the (16;16)(p13;q22) and del(16)(q22). The latter is associated with previous MDS, older age, a complex karyotype and a worse prognosis. Additional abnormalities include +8, +22, del(7q) and +2, which confer no change to the prognosis1.
The breakpoints occur in intron 5 of CBFB and intron 5 of MYH11. The N-terminal of CBFB fuses to the C-terminal of MYH11 with its multimerisation domain. The resultant chimaeric protein reduces the amount of active CBF. An accumulation of CBFB-MYH11/CBFA multimers in the nucleus also occurs. CBFB regulates expression of certain ADP-Ribosylation Factors (ARFs) and other tumour suppressor genes (TSGs) and therefore the fusion protein is thought to repress TSG expression3.
1. Huret, inv(16)(p13q22); t(16;16)(p13;q22); del(16)(q22). Atlas Genet Cytogenet Oncol Haematol 1999
2. Heim and Mitelman, Wiley-Liss Inc. 1995
3. Monreno-Miralles et al., J Biol Chem 2005;280(48):40097-103
Del (20q) Deletion
Deletions of the long arm of chromosome 20 are a common chromosomal abnormality associated with myeloid malignancies, in particular myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML)1. The deletions are predominantly interstitial2.
Often other cytogenetic abnormalities are present such as del(5q), -7/del(7q), +8, del(18q), +21 and rearrangements of 13q. Due to the relatively small size of the deletion and the lack of banding features on chromosome 20, FISH is particularly useful in detecting this abnormality. The prognosis for MDS cases where del(20q) is the sole abnormality is good. However if secondary abnormalities are present, a poor outcome is indicated3. AML patients respond poorly to treatment and have reduced survival rates. The clinical outcome for MPD patients remains unchanged in the presence of the abnormality4.
The mechanism of leukaemogenesis associated with del(20q) is unknown, however, deletion of a tumour suppressor gene (TSG) is thought to cause the increased proliferation of the cancer cells4. Using RT-PCR analysis, Bench et al identified potential target genes in the region of overlap between the AML/MDS and MPD CDR at band 20q12. Five genes were expressed in both bone marrow and CD34 positive cells. Of the three previously identified genes, L(3)MBT regulates chromatin structure during mitosis, SFRS6 encodes a serine rich protein important to regulation of alternative splicing of mRNA, and MYBL2, a member of the MYB transcription factor family, is involved in cell cycle control5,6,7.
1. Březinová et al., 2005:160(2):188-192
2. MacKinnon et al., Cancer Genet. 2011 Mar;204(3):153-61
3. Liu et al., Cancer Genet Cytogenet. 2006 Nov;171(1):9-16
4. Bilhou-Nabera, del(20q) in myeloid malignancies. Atlas Genet Cytogenet Oncol Haematol 2000
5. Bench et al., Oncogene 2000;19(34):3902-13 6. Li J et al., PNAS 2004;101:7341-67. Wang et al., Genomics 1999;59:275-81
This product is intended to be used on Carnoy’s solution (3:1 methanol/acetic acid) fixed haematological samples.
*Disease information supported by the literature and is not a reflection of the intended purpose of this product.