Background:
The CBFB::MYH11 gene fusion, derived from inv(16) or t(16;16) chromosomal rearrangement, is one of the two defining gene fusions for core binding factor acute myeloid leukemia (CBF AML). CBF AML harboring a CBFB::MYH11 gene fusion accounts for 5-7% of all AML cases (PMID: 23160462) and is recognized as a unique diagnosis with relatively favorable prognosis by both ICC and WHO classifications (PMID: 34135311; PMID: 22180162, PMID: 9746770, PMID: 25111512; PMID: 26660431; PMID: 27276561; PMID: 27895058).
CBF AMLs are defined by chromosomal translocations involving components of the CBF transcription factor complex, an important master transcriptional regulator of adult hematopoiesis. The CBF complex is comprised of two units: (1) a DNA-binding CBFα subunit (RUNX1, RUNX2, or RUNX3), and (2) a non-DNA-binding heterodimerization partner CBFβ subunit (encoded by the CBFB gene). Wild-type CBFB is a component of the CBFβ subunit; CBFB::MYH11 gene fusions disrupt the normal function of this transcriptional complex resulting dysregulation of myeloid cell growth and differentiation (PMID: 27798625; PMID: 32929473).
Clinical Utility:
This assay is intended to detect the most common type A CBFB::MYH11 chimeric RNA transcript (CBFB exon 5 joined to MYH11 exon 34) for either the initial diagnosis of a CBF leukemia and/or the continued monitoring of measurable residual disease in treated CBF AML patients. The 2021 ELN measurable residual disease (MRD) working party recommends CBF AML MRD assessment by qPCR or dPCR at the following intervals: (1) in PB after 2 cycles of chemotherapy, (2) in BM at the end of consolidation treatment, and (3) in PB every 4 to 6 wk for 24 mo after the end of consolidation. In the AML-15 trial, MRD monitoring by quantitative RT-PCR in CBF AML allowed risk stratification based on treatment responses after induction and consolidation chemotherapy and sequential monitoring during follow-up accurately predicted relapse (PMID: 22875911).
Methodology:
This reverse transcription digital polymerase chain reaction (RT-dPCR) assay detects CBFB::MYH11 chimeric RNA transcripts with a sensitivity of 10^-5. This assay detects only the most frequently observed type A CBFB::MYH11 breakpoint, estimated to represent 86% of total CBFB::MYH11 fusions:
- transcript type A fuses CBFB exon 5 (NM_001755.2) to MYH11 exon 34 (NM_001040113.2) (chr16: 67116211 - chr16: 15814908)
The other less common CBFB::MYH11 breakpoints, each estimated to represent 5% of total CBFB::MYH11 fusions, will not be detected by this assay:
- transcript type D fuses CBFB exon 5 (NM_001755.2) to MYH11 exon 30 (NM_001040113.2) (chr16: 67116211 - chr16: 15818849), and
- transcript type E fuses CBFB exon 5 (NM_001755.2) to MYH11 exon 29 (NM_001040113.2) (chr16: 67116211 - chr16: 15820911).
CBFB::MYH11 fusion transcript abundance is expressed as a normalized copy ratio of fusion transcripts relative to copies of ABL1 transcripts.
Total RNA extracted from fresh blood or bone marrow is submitted to a one-step RT-dPCR protocol, wherein the reverse transcription and digital PCR reactions are performed in the same well of a digital PCR nano-plate. Analyte is quantitively detected by the QIAcuity One 5-plex digital PCR instrument that simultaneously assesses the presence of amplified PCR product in approximately 25,000 micro-fluidically partitioned PCR reactions, each containing approximately 20 nanoliters total volume.
Specimen Requirements:
Blood: EDTA or ACD (Solution A or B):
- Adult: 5-10mL
- Child: 5mL
- Infant: 2-3mL
Bone Marrow: EDTA or ACD (Solution A or B):
- Adult: 5-10mL
- Child: 5mL
- Infant: 2-3mL
RNA: 10µg at a minimum of 100ng/µL (RNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS)
If the sample is drawn after treatment and lacks abundant blasts, the specific CBFB:: MYH11 fusion breakpoint from the pre-treatment diagnostic AML must be known, as defined by a molecular pathology report from a CLIA-certified laboratory. If the sample is bone marrow, a Pathology report MUST accompany the sample for accurate interpretation of results.
A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES. Please include detailed clinical information, including the known translocation breakpoint, ethnicity, clinical history, and family history.
Test Performed (Days):
Weekly
Turn Around Time:
7-10 days
Shipment Sensitivity Requirements:
- Package blood and bone marrow specimens to remain cold, but not frozen in transport.
- RNA must be stored and shipped frozen.
- Ship via overnight express, using the FedEx priority overnight label provided.
- Contact Client Services for shipping kits and instructions at (855) 535-1522.
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Additional Info: