Fluorescence in situ hybridization (FISH) is a technique that utilizes hybridization of fluorescence labeled DNA probes to specific chromosomal regions to detect specific chromosome abnormalities. The abnormalities may be translocations, deletions, inversions, trisomies or amplification in the interphase nuclei.

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Indications:

  1. Rapid detection of specific translocations
  2. Gene amplification can be detected
  3. Confirmation of rearrangements difficult to detect on karyotyping
  4. Results can be obtained in absence of dividing cells following treatment
  5. Detection of an early relapse
Several recurrent balanced translocations and inversions, and their variants, are recognized in the WHO category acute myeloid leukemia (AML) with recurrent genetic abnormalities, of which common ones can be detected using the following probes:

FISH for PML-RARA detection [t(15;17)]

  1. AML M3 and AML M3v are characterized by a reciprocal translocation between the long arm of chromosome 15 and the long arm of chromosome 17. This translocation leads to a rearrangement of the PML gene situated on chromosomal band 15q24 and the RARA gene situated on band 17q21. The PML-RARA rearrangement has gained major clinical importance because, in combination with all-trans retinoic acid (ATRA) and conventional anthracycline and cytarabine based chemotherapy, it leads to an improved prognosis in this subgroup of AML.
  2. XL PML/RARA probe is designed as a dual fusion probe. An orange labeled probe spans the breakpoint at 15q24 (PML) and a green labeled probe the breakpoint at 17q21 (RARA).

FISH for AML1/ ETO(RUNX1-RUNX1T1) [t(8;21)]

  1. The RUNX1 gene, located on chromosome 21q22.1, is crucial for the establishment of definite haematopoiesis and the generation of hematopoietic stem cells in the embryo. The most common translocations involving RUNX1 are the t(8;21) RUNX1T1/RUNX1 in AML and t(12;21) ETV6/RUNX1 in acute lymphoblastic leukemia (ALL), both associated with a more favorable diagnosis.
  2. The XL AML1/ETO is designed as a dual fusion probe. The orange labeled probes flank the breakpoint at 21q22.1 (AML1=RUNX1), and the green labeled probes flank the breakpoint at 8q21(ETO=RUNX1T1).

FISH for MLL/KMT2A (break apart)

  1. The KMT2A (formerly MLL) gene, located on chromosome 11q23, is rearranged in about 10% of all acute leukemia patients. In infants, the incidence of KMT2A rearrangements in leukemia is 70-80%.
  2. The XL MLL plus probe is designed as a break-apart probe for gene locus 11q23. Its orange labeled part hybridizes proximal to the gene locus i.e. at D11S4195 AND TMPRSS4 at 11q23; the green labeled probe hybridizes to the distal region i.e. at DDX6 AND D11S4538 at 11q23.

FISH for inv(16) [CBFB-MYH11]

  1. In cases with inv(16)/t(16;16), the core binding factor b (CBFB) gene on 16q22 is fused with the smooth muscle myosin heavy chain gene (MYH11) on 16p13.1. These recurrent rearrangements are present in about 10% of young AML patients.
  2. The XL CBFB/MYH11 probe is designed as a dual fusion probe. An orange labeled probe spans the breakpoint at 16q22 and includes the CBFB locus. A green labeled probe spans the breakpoint at 16p13 and includes the MYH11 locus.
It is an aggressive type of leukemia which involves uncontrolled growth of B-cell lymphoblasts in the bone marrow and blood. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer type. Genomic data suggests that more than 10 functional aberrations contribute to the development of this disease.

FISH for BCR-ABL1 [t(9;22)]

  1. The t(9;22)(q34;q11) translocation results in a BCR-ABL1 gene fusion on the derivative chromosome 22, called the Philadelphia (Ph) chromosome.
  2. BCR-ABL1 fusion is found in chronic myeloid leukemia, BCR-ABL1 positive, some cases of B-lymphoblastic leukemia, acute myeloid leukemia(~1%) and mixed phenotypic acute leukemia, BCR-ABL1 positive.
  3. The XL BCR/ABL1 plus probe is specific for the t(9;22). The orange labeled probe detects an extended region at the ABL1 locus on 9q34, and a green labeled probe hybridizes specifically to regions at the BCR gene on 22q11. The probe is designed as a dual-color, dual-fusion assay.

FISH for MLL/KMT2A (break apart)

  1. The KMT2A (formerly MLL) gene, located on chromosome 11q23, is rearranged in about 10% of all acute leukemia patients. In infants, the incidence of KMT2A rearrangements in leukemia is 70-80%.
  2. The XL MLL plus probe is designed as a break-apart probe for gene locus 11q23. Its orange labeled part hybridizes proximal to the gene locus i.e. at D11S4195 AND TMPRSS4 at 11q23; the green labeled probe hybridizes to the distal region i.e. at DDX6 AND D11S4538 at 11q23.

FISH for TEL- AML1 [t(12;21), ETV6/RUNX1]

  1. The most common aberration in pediatric B-cell ALL is t(12;21)(p13;q22) with an incidence of about 25%, compared to < 5% in adults. The result of this reciprocal translocation is an ETV6-RUNX1 fusion gene.
  2. The XL t(12;21) ETV6-RUNX1 is designed as a dual fusion probe. The green labeled probes flank the breakpoint at 12p13 (TEL=ETV6), and the orange labeled probes flank the breakpoint at 21q22 (AML1=RUNX1).

FISH for E2A/TCF3 detection [t(1;19)]

  1. The t(1;19) is one of the most common chromosomal abnormalities in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The TCF3 gene has been shown to be involved in the majority of cases with a cytogenetically visible t(1;19) translocation.
  2. The XL E2A probe is designed as a break-apart probe with two probes juxtaposed and differently labeled, for locus 19p13 (E2A=TCF3). The proximal probe i.e. RH98588 is labeled in orange, and a green labeled probe is designed to hybridize distal region i.e. D19S883.
The prognosis and clinical course of chronic lymphocytic leukemia (CLL) are heterogeneous. With FISH, abnormalities can be detected in more than 80% of patients by using a 4-probe panel for the detection of trisomy 12, and deletions 13q, 17p, and 11q.

FISH for 11q (ATM)

  1. Chromosome 11q22.3-23.1 deletions involving the ataxia telangiectasia mutated (ATM) locus are detected at diagnosis in 15 – 20% of cases of B-cell CLL and are associated with a more aggressive disease.

FISH for 17p (p53)

  1. TP53 is a tumor suppressor gene which stops cell division when DNA is damaged. Loss of TP53 at 17p13 is a powerful predictor of resistance to therapy with purine analogues and alkylating agents, and of poor prognosis in CLL.
  2. The XL ATM/TP53 locus-specific probe detects deletions in the long arm of chromosome 11 and in the short arm of chromosome 17. The green labeled probe detects a specific region at 11q22 covering the ATM gene. The orange labeled probe hybridizes specifically to the TP53 gene at 17p13.

FISH for del(13q)

  1. The most frequently deleted region in B-CLL is located in 13q14.3 distal to RB1. CLL with 13q deletion as the sole cytogenetic abnormality usually has a good prognosis.
  2. The XL DLEU/LAMP/12cen is a 3-color probe that hybridizes to the DLEU gene region at 13q14 (including the D13S319 marker) in orange, the LAMP gene region at 13q34 in blue (aqua), and a chromosome 12 centromeric probe labeled in green.

FISH for Trisomy 12

  1. Trisomy 12 is present in approximately 16% of cases of CLL. However, the prevalence of this cytogenetic abnormality is significantly higher in small lymphocytic lymphoma (SLL) where it is present in 28% of cases.
The most common MM-associated IGH translocations are t(11;14), t(4;14), t(6;14), t(14;16) and t(14;20) in the order of their occurrence. The consequence of these rearrangements is the dysregulation of genes juxtaposed to transcriptional enhancers in the IGH locus. Prognosis and risk stratification strongly depend on the detection and interpretation of cytogenetic primary abnormalities. t(14;16) and t(14;20) are considered as high risk, t(4;14) as intermediate risk and t(6;14) and t(11;14) as standard risk cytogenetic aberrations in patients with MM based on FISH testing. Plasma cells are isolated from a bone marrow (BM) aspirate using CD138+ Microbeads (Miltenyi biotech). CD138+ cells (plasma cells) are then analyzed by FISH using specific probes.

FISH for 11q (ATM) and 17p (p53) deletion

  1. Deletion of chromosome 17p13 [del(17p)] is found in ~10% of newly diagnosed (ND) MM and at higher prevalence in advanced disease. The XL ATM/TP53 locus-specific probe detects deletions in the long arm of chromosome 11 and in the short arm of chromosome 17. The green labeled probe detects a specific region at 11q22 covering the ATM gene. The orange labeled probe hybridizes specifically to the TP53 gene at 17p13.

FISH for DLEU/ LAMP/ 12cen

  1. The XL DLEU/LAMP/12cen is a 3-color probe that hybridizes to the DLEU gene region at 13q14 (including the D13S319 marker) in orange, the LAMP gene region at 13q34 in blue (aqua), and a chromosome 12 centromeric probe labeled in green.

FISH for IgH

  1. Chromosomal translocations affecting the immunoglobulin heavy chain (IGH) locus at 14q32.3 are recurrent in many types of lymphomas and plasma cell neoplasms. The consequence of these rearrangements is the dysregulation of genes juxtaposed to transcriptional enhancers in the IGH locus. Due to the telomeric position of the IGH locus, 14q32.3 translocations may be easily missed by conventional cytogenetics. FISH is therefore a valuable tool in the diagnosis of translocations affecting the IGH locus.
  2. The XL IGH BA consists of an orange-labeled probe partly covering the constant region of the IGH locus at 14q32.3 and a green-labeled probe hybridizing to the variable distal region of the IGH locus at 14q32.3.

FISH for t(11;14) MYEOV/IGH

  1. In multiple myeloma (MM), t(11;14) is the most common translocation, detectable by FISH in about 15-20% of all MM patients. Conventional cytogenetics has a much lower sensitivity, detecting t(11;14) in about 5% of MM patients.
  2. The XL t(11;14) MYEOV/IGH DF consists of an orange-labeled probe hybridizing to the MYEOV/CCND1 gene region at 11q13.3 and a green-labeled probe hybridizing to the IGH gene region at 14q32.3.

FISH for t(4;14) FGFR3/IGH

  1. The recurrent translocation t(4;14)(p16;q32) juxtaposes FGFR3 with the 3′ alpha IgH enhancer on der(14), whereas expression of NSD2 is controlled by the Eμ enhancer on der(4).
  2. XL t(4;14) FGFR3/IGH DF consists of an orange-labeled probe hybridizing to the FGFR3 gene region at 4p16.3 and a green-labeled probe hybridizing to the IGH gene region at 14q32.3.

FISH for t(14;16) IGH/MAF

  1. MAF overexpression caused by t(14;16)(q32;q23) increases gene expression levels of the downstream target genes cyclin D2 and integrin beta 7 and contributes to the pathogenesis of MM.
  2. XL t(14;16) IGH/MAF DF consists of a green-labeled probe hybridizing to the IGH gene region at 14q32.3 and an orange-labeled probe hybridizing to the WWOX/MAF gene region at 16q23.

FISH for 1p Deletion/1q Amplification

  1. Deletions of chromosome 1p have been described in 7-15% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. Gain of 1q is one of the most recurrent chromosomal aberrations in MM. Amplification and overexpression of the CKS1B gene in chromosome band 1q21 has been associated with an aggressive clinical course in MM.
  2. The XL CDKN2C/CKS1B consists of a green-labeled probe hybridizing to the CDKN2C (p18) gene region at 1p32.3 and an orange-labeled probe hybridizing to the CKS1B gene region at 1q21-22.

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders associated with ineffective hematopoiesis and peripheral blood cytopenias. MDS patients have a significant risk of progression to acute myeloid leukemia. MDS are associated with deregulated production of myeloid cells. According to WHO classification (2008), cytogenetic aberrations are observed in about 50% of MDS cases. The most common aberrations are 5q-, -7/7q-, trisomy 8, del(20q).

FISH for del(5q) [5q31/5q33/5p15]

  1. 5q syndrome is defined as a primary MDS with del(5q) as the sole karyotypic abnormality. Two different critical regions are described, one is located at 5q31 and contains the EGR1 and CDC25C genes. A more distal region at 5q32-q33 containing RPS14 has been identified as a causal region for 5q syndrome.
  2. The XL 5q31/5q33/5p15 locus-specific probe detects deletions in the long arm of chromosome 5. The orange labeled probe hybridizes to a specific region at 5q31 including the EGR1 gene. The green labeled probe hybridizes to 5q33 and includes the RPS14 gene. A blue (aqua) labeled probe hybridizes to 5p15 as a control.

FISH for del(7q) [7q22/7q36]

  1. Loss of chromosome 7 (-7) or deletion of the long arm (7q-) are recurrent chromosome abnormalities in myeloid leukemias such as MDS or acute myeloid leukemia (AML). The association of -7/7q with myeloid leukemia suggests that certain regions contain tumor suppressor gene(s), whose loss of function contribute to leukemic transformation or tumor progression. Multiple critical regions have been identified: one in band 7q22 (including the KMT2E gene), the more telomeric region 7q31 and 7q35-q36 (including the EZH2 gene).
  2. The XL 7q22/7q36 locus-specific probe detects deletions in the long arm of chromosome 7. The orange labeled probe hybridizes to a specific region at 7q22 including the KMT2E (formerly MLL5) gene. The green labeled probe hybridizes specifically to 7q36 and includes the EZH2 gene. A blue (aqua) labeled probe which hybridizes to the centromere of chromosome 7 functions as a reference probe.

FISH for 20q del

  1. Del(20q) is a recurrent but rare aberration and is present in about 3-7% of MDS patients. The majority of cases have an interstitial deletion which is flanked by the PTPRT gene and includes the MYBL2 gene. Sole del(20q) is associated with a more favorable outcome.
  2. The XL Del(20q) detects deletions which occur in the long arm of chromosome 20. The probe mixture includes two probes: an orange labeled one which hybridizes to 20q12 and includes the proximal part of PTPRT, and a green labeled probe which hybridizes to 20q13 and includes the MYBL2 locus.
Follicular lymphoma (FL) is typically a slow-growing or indolent form of non-Hodgkin lymphoma (NHL) that arises from B-lymphocytes. This lymphoma subtype accounts for 20 to 30 percent of all NHL cases.

FISH for IGH/BCL2 [t(14;18)]

  1. Rearrangements of the immunoglobulin heavy chain (IGH) gene locus are present in about 50% of all non-Hodgkin lymphomas (NHL) including follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL). The reciprocal translocation t(14;18)(q32;q21) can be detected in about 85% of FL patients and in up to 35% of patients with DLBCL.
  2. Rearrangements of the immunoglobulin heavy chain (IGH) gene locus are present in about 50% of all non-Hodgkin lymphomas (NHL) including follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL). The reciprocal translocation t(14;18)(q32;q21) can be detected in about 85% of FL patients and in up to 35% of patients with DLBCL.
Mantle cell lymphoma is typically an aggressive, rare, form of non-Hodgkin lymphoma (NHL) that arises from cells originating in the “mantle zone.” Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) with an aggressive clinical course.

FISH for MYEOV/IGH [t(11;14)]

  1. It is genetically characterized by t(11;14)(q13;q32) which is present in about 95% of MCL patients.
  2. The XL t(11;14) MYEOV/IGH DF is designed as a dual fusion probe. The orange labeled probe hybridizes to gene region 11q13 (CCND1/MYEOV), the green labeled probe flanks the IGH breakpoint region at 14q32.
DLBCL is an aggressive (fast-growing) NHL that affects B-lymphocytes. Although it can occur in childhood, the occurrence of DLBCL generally increases with age, and most patients are over the age of 60 at diagnosis. Diffuse large B-cell lymphoma (DLBL) represents one of the most common entities of non-Hodgkin lymphoma (NHL) worldwide.

FISH for BCL6

  1. In DLBLs, a BCL6 translocation can be found in up to 40% of cases.
  2. The XL BCL6 BA consists of a green-labeled probe hybridizing proximal to the BCL6 gene region at 3q27 and an orange-labeled probe hybridizing distal to the BCL6 gene region at 3q27-28.

FISH for MYC

  1. Translocations involving MYC are observed in diffuse large-B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and other lymphomas.
  2. The XL MYC BA probe is designed as a break apart probe. The orange labeled probe hybridizes proximal to the breakpoint in the MYC gene region at 8q24, the green labeled probe hybridizes distal to the breakpoint.

FISH for IGH/BCL2 [t(14;18)]

  1. The reciprocal translocation t(14;18)(q32;q21) can be detected in up to 35% of patients with DLBCL.
  2. t(14;18) IGH/BCL2 DF consists of a green-labeled probe hybridizing to the IGH gene region at 14q32.3 and an orange-labeled probe hybridizing to the BCL2 gene region at 18q21.3.
Neoplasms with eosinophilia are associated with dysregulated tyrosine kinases, usually as a result of gene fusions. It is of great importance to identify the genes involved because aberrant tyrosine kinases react with varying sensitivity to tyrosine kinase inhibitors. The updated (2016) World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues indicates the category myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, FGFR1, or with PCM1-JAK2. Patients with PDGFRA and PDGFRB rearrangements are responsive to imatinib.

FISH for PDGFR ALPHA

  1. The most frequent PDGFRA-related aberration is the interstitial deletion of the CHIC2 gene with breakpoints in the FIP1L1 and PDGFRA genes. In FISH assays, the detection of CHIC2 deletion at 4q12 is a surrogate for the direct detection of the FIP1L1-PDGFRA fusion gene.
  2. The XL PDGFRA BA consists of a green-labeled probe hybridizing proximal to the PDGFRA gene region at 4q12 and an orange-labeled probe hybridizing distal to the PDGFRA gene region at 4q12.

FISH for PDGFR BETA

  1. Myeloid neoplasms (MPNs) with rearrangement of PDGFRB are phenotypically and genotypically diverse. The fusion genes involving PDGFRB described to date have been associated with cytogenetically detectable translocations. MPNs associated with rearrangement of PDGFRB are responsive to imatinib.
  2. The XL 5q32 PDGFRB BA consists of an orange-labeled probe hybridizing proximal to the PDGFRB gene region at 5q32 and a green-labeled probe hybridizing distal to the PDGFRB gene region at 5q32-33.1.

FISH for Trisomy 8

  1. Trisomy 8 is a recurrent, non-random abnormality seen primarily in myeloid disorders, including myelodysplasia, myeloproliferative neoplasms and acute myeloid leukemia and also seen in CML as a major additional secondary change with the t(9;22).
  2. Trisomy 8 is found in 10–15% of AML and 10% of treatment-related AML. It is present in each FAB subgroup, and many cases may arise from a preceding MDS. Trisomy 8 as the sole anomaly in AML is seen in 40% of cases, with the remaining cases seen with other cytogenetic changes including del(5q), del(7q), monosomy 5 and 7, often associated with complex karyotype anomalies. The prognosis of AML in adults with trisomy 8 is generally associated with an intermediate to poor prognosis.

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