Case ReportCharacterizing false-positive fluorescence in situ hybridization results by mate-pair sequencing in a patient with chronic myeloid leukemia and progression to myeloid blast crisis
Introduction
Hematopoietic and lymphoid neoplasms are often characterized by specific cytogenetic and molecular abnormalities that determine diagnosis, prognosis and in some cases guide therapy-related decisions [1]. Conventional chromosome analysis is well suited to detect abnormalities such aneuploidies, large gains and/or losses (~5–10 Mb) and balanced rearrangements observed in individual cells [2]. Fluorescence in situ hybridization (FISH) has a resolution of ~100kb-1Mb and is considered a highly sensitive and relatively rapid technique that is particularly useful in identifying targeted, disease specific abnormalities that may be unappreciated by conventional chromosome analysis. In addition, FISH studies can be performed on a large number of non-dividing interphase cells [3]. However, with the advent of novel next-generation sequencing (NGS) techniques, including mate-pair sequencing (MPseq), apparent gene fusions or rearrangements predicted by chromosomes and/or FISH studies have been disproven on the DNA level [4,5].
We present a 27-year-old male with a history of chronic myeloid leukemia (CML) and progression to myeloid blast crisis and a complex karyotype obtained by a peripheral blood specimen, including t(9;22)(q34;q11.2) and t(4;11)(q12;p15). Suspicious of a novel NUP98/PDGFRA gene fusion resulting from the t(4;11)(q12;p15), break-apart (BAP) FISH probes were performed for each gene region and demonstrated FISH signal patterns consistent with balanced PDGFRA and NUP98 gene rearrangements. MPseq was subsequently performed for confirmatory purposes and verified the BCR/ABL1 fusion associated with the t(9;22)(q34;q11.2). However, while MPseq confirmed a t(4;11)(q12;p15) and involvement of the NUP98 gene at 11p15 (intron 12, NM_01620), an intergenic region was identified at the 4q12 breakpoint, indicating this translocation does not create the predicted NUP98/PDGFRA gene fusion event.
Section snippets
Case presentation
A peripheral blood specimen was sent to the Mayo Clinic genomics laboratory for acute myeloid leukemia (AML) FISH studies and conventional chromosome analysis from a 27-year-old male with a previous diagnosis of CML and current progression to myeloid blast crisis. A complete blood count revealed anemia (hemoglobin, 10.9 g/dL; reference, 14–18 g/dL), thrombocytopenia (platelet count, 40 K/μL; reference, 150–350 K/μL), and leukocytosis [white blood cell (WBC) count, 148.4 K/μL; reference,
Conventional chromosome analysis
All 20 metaphases evaluated from the peripheral blood study had a complex karyotype resulting in the flowing composite karyotype: 43–47,XY,t(4;11)(q12;p15),inv(5) (p13q13)?c,+8,t(9;22)(q34;q11.2),add(16)(q13),+0~1mar[cp20] (Fig. 1A).
Fluorescence in situ hybridization
FISH studies identified a balanced d-FISH signal pattern demonstrating BCR/ABL1 fusion (Fig. 1B). In addition, FISH studies also identified apparently balanced BAP FISH signal patterns indicating a NUP98 rearrangement (Fig. 1C) and a PDGFRA rearrangement (Fig. 1D)
Discussion
Herein we have utilized MPseq to characterize the precise breakpoints for an apparently novel t(4;11)(q12;p15) identified in a 27-year-old patient with CML in myeloid blast crisis and predicted by BAP FISH testing to involve the PDGFRA and NUP98 gene regions. The molecular characterization of the 4;11 translocation indicated a false-positive PDGFRA FISH result and the lack of the predicted PDGFRA/NUP98 fusion event.
The nucleoporin 98 (NUP98) gene located at 11p15.4 has been associated with at
Conclusion
While traditional cytogenetic techniques have greatly advanced our understanding of hematologic neoplasms, newer NGS-based technologies, including MPseq, have surpassed the resolution afforded by chromosome and FISH methodologies. Lastly, while MPseq may not supplant the routine use of traditional cytogenetic methodologies, this novel NGS-based approach can be beneficial for the characterization of complex rearrangements or potential novel gene fusions.
Declaration of Competing Interest
None
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