Test Directory

• PML::RARA, t(15;17)
• CBFB::MYH11, inv(16)
• MLL (KMT2A) (11q23.3) Rearrangement
• AML1::ETO, RUNX1::RUNX1TA, t(8;21)
• RARA (17q21.1-q21.2) Rearrangement
• TP53 (17p13)/17cen Deletion
• ETV6 (12p13) Rearrangement
• MECOM (EVI1) (3q26.2) Rearrangement

PML::RARA, t(15;17)(q24;q21) Translocation Probe

The genetic hallmark of acute promyelocytic leukemia (APL) is a balanced chromosomal translocation t(15;17)(q24;q21) leading to fusion of the promyelocytic leukemia (PML) and the retinoic acid receptor alpha (RARA) genes. The fusion gene results in a chimeric PML::RARA oncoprotein, which plays a causative role in APL pathogenesis impairing myeloid differentiation. The resulting PML::RARA protein silences genes that are critical for cell differentiation, resulting in both a differentiation block and an expansion of myeloid progenitors. Acute promyelocytic leukemia (APL) accounts for 10% of adult myeloid leukemia.

Although all patients with APL have t(15;17) or a variant of this translocation, conventional cytogenetic methods detect these translocations in only 70–96% of patients at diagnosis. FISH for abnormalities of chromosome PML::RARA can be performed on interphase or metaphase cells.

CBFB::MYH11, inv(16)(p13q22) Translocation Probe

Acute myeloid leukaemia with CBFB::MYH11 fusion is characterized by fusion of the core-binding factor subunit beta (CBFB) and the myosin heavy chain 11 (MYH11) genes. This AML type comprises in 5-8% of cases in younger adult patients and decreases in incidence in older adults. The most common cytogenetic alteration, inv(16)(p13.1q22.1), detected in ~95% of patients, may be difficult to identify on conventional karyotyping in some cases. Secondary chromosomal aberrations are present in ~45% of AML with CBFB::MYH11 and include trisomies 8 (10-20%), 21 (~5%) and 22 (15-25%), as well as del(7q) (~5%). Somatic mutations are detected in >90% of cases and commonly involve NRAS (30-50%) and KRAS (10-15%), followed by KIT, ASXL2, ASXL1, NF1, TET2, EZH2, FLT3 tyrosine kinase domain, and FLT3 internal tandem duplication. The prognosis of AML with CBFB::MYH11 is favorable, with long-term survival rates of ~50% in adults. FISH for abnormalities of chromosome CBFB::MYH11, inv(16) can be performed on interphase or metaphase cells.

MLL (KMT2A), 11q23 Breakapart Probe-Rearrangement

Mixed lineage leukemia (MLL) abnormalities are frequently found in infant leukemias and therapy-related leukemias. Acute myeloid leukemia with KMT2A rearrangement is a myeloid neoplasm in which the KMT2A locus on chromosome 11q23.3 is fused with any of a number of partner genes. KMT2A gene rearrangements result from deletions, duplications, inversions and translocations at the 11q23 locus. More than 80 fusion partners have been described in KMT2A rearranged acute myeloid leukemia (AML). Overall, MLLT3, AFDN, ELL, and MLLT10 are most commonly implicated in KMT2A-rearranged AML, resulting from the translocations t(9;11)(p22;q23), t(6;11)(q27;q23), t(11;19)(p13.1;q23), and t(10;11)(p12;q23), respectively. As compared to AML with normal karyotype, the presence of KMT2A rearrangement is associated with a lower mutation burden . Adult patients predominantly have mutations in genes encoding the RAS pathway (NRAS, KRAS and PTPN11). FLT3 tyrosine kinase domain mutations (TKD), as well as mutations in RUNX1, TET2, PLCG2 and ZRSR2, have also been described. Mutations in NRAS, KRAS, PTPN11 and FLT3-TKD often occur at low (<20%) variant allele fractions indicating a subclonal nature. Mutations in DNMT3A, NPM1, CEBPA and FLT3 internal tandem duplication are uncommon. FISH can detect this abnormality in either interphase or metaphase cells.

AML1 (RUNX1)::ETO (RUNX1T1), t(8;21) Translocation Probe

The t(8;21)(q22;q22.1) is found in 1-5% of AML. RUNX1::RUNX1T1 is the predominant core binding factor (CBF) rearrangement in African American AML patients (~55-70% of CBF AML). The presence of KIT p.D816 in adults correlates with a lower relapse-free survival rate, while hyperdiploidy and/or presence of del(9q) is associated with longer overall survival. A high (≥2) mutation burden is associated with inferior outcomes.

ETV6, 12p13 Breakapart Probe-Rearrangement

Deregulation of the ETV6 gene through translocation, deletion or somatic mutation is recurrent in myeloid malignancies. By metaphase cytogenetics (MC), approximately 1% of patients with AML and 0.6% of patients with MDS have deletion of part of 12p13.2 Mapping studies place ETV6 within the smallest commonly deleted region.

EVI1 (MECOM), 3q26.2 Breakapart Probe-Rearrangement:

The MECOM [myelodysplasia syndrome 1 (MDS1) and ecotropic virus integration site 1 protein homolog (EVI1) complex locus] oncogene at 3q26.2 is often rearranged in hematological malignancies of myeloid origin. MECOM encodes a zinc finger protein that is inappropriately expressed in the leukemic cells of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. This deregulated expression is often due to a chromosomal rearrangement involving 3q26.2, with the two most common aberrations being the t(3;3)(q21;q26.2) and inv(3)(q21q26.2). The breakpoints for the translocations and inversions vary considerably. Inversion breakpoints are found centromeric to, and include the MECOM gene, covering about 600kb. The majority of breakpoints in 3q26.2 translocations are telomeric to the MECOM gene and cover a region including the telomeric end of the MDS1 gene and the MYNN gene2. Chromosome rearrangements involving the 3q26.2 region are associated with myeloid malignancies, aberrant expression of MECOM gene and an unfavorable prognosis2. AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) is a recognized disease entity according to the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. This is a transformed or de novo AML with a very aggressive clinical course and aberrations that involve MECOM at 3q26.2 and RPN1 (ribophorin I) at 3q213. MECOM has also been shown to be rearranged in therapy-related disease via the t(3;21)(q26.2;q22) translocation, resulting in a MECOM-RUNX1 fusion3,4. MECOM rearrangements are very heterogeneous and may be difficult to detect by conventional cytogenetics, making FISH a useful tool for their detection.

RARA, 17q21.1-q21.2 Breakapart Probe-Rearrangement

FISH: Retinoic acid receptor alpha (RARA) is a dual color break apart test that is performed to detect the molecular rearrangement RARA (17q21.1). This FISH assay is used in the diagnosis of acute promyelocytic leukemia (APL).

TP53, 17p13/17cen, 17p11.1-q11.1 Deletion Probe

The tumor protein 53 (P53, TP53) Deletion FISH Probe Kit is a fluorescence in situ hybridization (FISH) Test used to detect deletion of the P53 (TP53) region on chromosome 17 at location 17p13 in fixed bone marrow specimens from patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The test is indicated under revised diagnostic criteria for myeloid neoplasms (MN) issued by the International Consensus Classification (ICC) and the World Health Organization (WHO) recommended major change pertaining to TP53-mutated (TP53mut) MN, and in conjunction with other clinicopathological criteria.

Specimen Types: Leukemic Blood or Bone Marrow

Preferred Collection Container: Green Top (Sodium Heparin)

Alternate Collection Container: Lavendar Top (EDTA)

Desired Specimen Volume: 3 - 5mL

Minimum Acceptable Volume: 1 mL

• Lithium heparin is not acceptable.
• Specimens must be collected in a sterile collection container.

Each specimen must be accompanied by a requisition form, script, electronic order or physician note. Each order must indicate the reason for testing (diagnosis).

• Transport to the laboratory at room temperature.
• Store specimen as whole blood, in original tube, at room temperature and deliver to lab within 24 hours.
• If specimen cannot be delivered within 24 hours, store in refrigerator. Do not freeze specimen.
• Specimens collected on the weekends must be refrigerated upon arrival at HNL Lab Medicine.

• Specimen frozen
• Specimen arriving without any form of patient identification (unlabeled)
• Specimen with attached needles
• Specimen arriving with broken or leaking collection tube
• Unfixed specimen treated with fixative

PML::RARA, t(15;17)(q24;q21) Translocation Probe

In a normal cell, the expected signal pattern is two red signals and two green signals. In an abnormal cell containing a typical translocation, the expected signal pattern is one red signal, one green signal, and two fused signals.

CBFB::MYH11, inv(16)(p13q22) Translocation Probe

In a normal cell, the expected signal pattern is two red signals and two green signals. In an abnormal cell containing a typical translocation, the expected signal pattern is one red signal, one green signal, and two fused signals.

KMT2A (MLL), 11q23 Breakapart Probe-Rearrangement

In a normal cell, the expected signal pattern is two fused signals. In an abnormal cell with a rearrangement, the expected signal pattern is one red signal, one green signal, and one fused signal.

AML1(RUNX1)::ETO(RUNX1T1), t(8;21) Translocation Probe

In a normal cell, the expected signal pattern is two red signals and two green signals. In an abnormal cell containing a typical translocation, the expected signal pattern is one red signal, one green signal, and two fused signals.

RARA, 17q21.1-q21.2 Breakapart Probe-Rearrangement

In a normal cell, the expected signal pattern is two fused signals. In an abnormal cell with a rearrangement, the expected signal pattern is one red signal, one green signal, and one fused signal.

P53, 17p13/ 17cen Deletion Probe

In a normal cell, the expected signal pattern is two red signals and two green signals. In an abnormal cell containing the deletion, the expected signal pattern is one red signal and two green signals or one red signal and one green signal.

ETV6, 12p13 Breakapart Probe- Rearrangement,

In a normal cell, the expected signal pattern is two fused signals. In an abnormal cell with a rearrangement, the expected signal pattern is one red signal, one green signal, and one fused signal.

MECOM (EVI1), 3q26.2 Breakapart Probe- Rearrangement,

In a normal cell, the expected signal pattern is two fused signals. In an abnormal cell with rearrangement, the expected signal pattern is 1F(AGR)1F(GR)1A for inversion and 1F(AGR)1F(AG)1R for translocation.

• FISH AML Panel
• AML