CBFB Break-Apart FISH Testing: An Analysis of 1629 AML Cases with a Focus on Atypical Findings and Their Implications in Clinical Diagnosis and Management

Abstract

Fluorescence in situ hybridization (FISH) is a confirmatory test to establish a diagnosis of inv(16)/t(16;16) AML. However, incidental findings and their clinical diagnostic implication have not been systemically studied. We studied 1629 CBFB FISH cases performed in our institution, 262 (16.1%), 1234 (75.7%), and 133 (8.2%) were reported as positive, normal, and abnormal, respectively. The last included CBFB copy number changes (n = 120) and atypical findings such as 3'CBFB deletion (n = 11), 5'CBFB deletion (n = 1), and 5'CBFB gain (n = 1). Correlating with CBFB-MYH11 RT-PCR results, totally 271 CBFB rearrangement cases were identified, including five with discrepancies between FISH and RT-PCR due to new partner genes (n = 3), insertion (n = 1), or rare CBFB-MYH11 variant (n = 1) and eight with 3'CBFB deletion. All cases with atypical findings and/or discrepancies presented clinical diagnostic challenges. Correlating FISH signal patterns and karyotypes, additional chromosome 16 aberrations (AC16As) show impacts on the re-definition of a complex karyotype and prognostic prediction. The CBFB rearrangement but not all AC16As will be detected by NGS-based methods. Therefore, FISH testing is currently still needed to provide a quick and straightforward confirmatory inv(16)/t(16;16) AML diagnosis and additional information related to clinical management.

Keywords: CBFB rearrangement; CBFB-MYH11; FISH; RT-PCR; additional chromosome16 aberrations (AC16As); atypical findings; next-generation sequencing (NGS).

Figure 1

Schematic illustration of CBFB break-apart (BAP) and CBFB-MYH dual fusion (DF) FISH probe sets applied in this study. Information was obtained from the user’s guide provided by the manufacturers. (A). CBFB BAP probe set with coverages of 5′CBFB and flanking region (~130 kb) labeled with red dye, and 3′CBFB and flanking region (~204 kb) labeled with green dye. (B). CBFB-MYH11 DF probe set with coverages of CBFB and flanking region (~1270 kb) labeled with red dye, and MYH11 and flanking region (~1080 kb) labeled with green dye. The sizes are not to scale.

Figure 2

CBFB BAP FISH demonstrated that 3′CBFB signal (green) was translocated on an abnormal chromosome 1 (A). Case #1 and an abnormal chromosome 19 (B). Case #3, respectively. From left to right: metaphase FISH image with DAPI; inverted metaphase FISH image to show chromosome morphology; affected chromosomes and their normal homologs. Red: 5′CBFB; Green: 3′CBFB; Fusion (yellow): intact CBFB.

Figure 3

FISH studies with CBFB BAP FISH and CBFB-MYH11 DF FISH in two cases. (A). Case #4: Both BAP FISH (left, 1R1G1F signal pattern) and DF FISH (right, 1R1G2F) showed typical signal patterns for CBFB-MYH11 rearrangement. RT-PCR was negative in this case. (B). Case #5 showed a negative CBFB BAP FISH result (left). CBFB-MYH11 DF FISH indicated an insertion of MYH11 (green) into the CBFB (red), forming a fusion signal (right). RT-PCR was positive in this case.

Figure 4

Illustration of cryptic and complicated CBFB-MYH11 rearrangement in case #12: atypical FISH signal pattern (1R1F, A) by BAP FISH; one fusion signal (1R1G1F, B) by DF FISH. Whole chromosome 16 painting (wcp16) (C) excluded a possible recombination between one chromosome 16 and a non-16 chromosome.

Figure 5

Illustration of case #15 with inv(16) and an additional chromosomal 16 abnormality. An atypical FISH signal pattern (1R1F, A) was detected by BAP FISH. However, no CBFB-MYH11 fusion signal was detected by DF FISH (B), though the metaphase image indicated that the MYH11 was relocated on 16q (RT-PCR was also negative).