Elsevier

Cancer Genetics

Volume 243, May 2020, Pages 48-51
Cancer Genetics

Case Report
Characterizing false-positive fluorescence in situ hybridization results by mate-pair sequencing in a patient with chronic myeloid leukemia and progression to myeloid blast crisis

https://doi.org/10.1016/j.cancergen.2020.02.008Get rights and content

Highlights

  • Newly acquired clonal chromosomal abnormalities in chronic myeloid leukemia (CML) usually indicate disease progression.

  • Conventional chromosome and fluorescence in situ hybridization (FISH) studies detected t(4;11)(q12;p15) and NUP98 (11p15) and PDGFRA (4q12) rearrangements, respectively, in a patient with CML with progression to blast crisis.

  • A next generation sequencing assay, mate-pair sequencing (MPseq), did not detect the novel 5′NUP98/3′PDGFRA fusion as inferred by traditional cytogenetic methodologies, thus demonstrating the power of MPseq.

Abstract

Traditional cytogenetic testing methodologies, including conventional chromosome analysis and fluorescence in situ hybridization (FISH), are invaluable for the detection or recurrent genetic abnormalities in various hematologic malignancies. However, technological advances, including a novel next-generation sequencing technique termed mate-pair sequencing (MPseq), continue to revolutionize the field of cytogenetics by enabling the characterization of structural variants at a significantly higher resolution compared to traditional methodologies. To illustrate the power of MPseq, we present a 27-year-old male diagnosed with chronic myeloid leukemia in myeloid blast crisis with multiple chromosomal abnormalities observed in all 20 metaphases from a peripheral blood specimen, including t(9;22)(q34;q11.2) and t(4;11)(q12;p15). Suspicious of a novel NUP98/PDGFRA fusion [t(4;11)(q12;p15)], break-apart FISH probe sets for the PDGFRA (4q12) and NUP98 (11p15.4) gene regions were performed and were both positive in approximately 86% of 200 interphase nuclei. However, subsequent MPseq testing revealed breakpoints located within the NUP98 gene and within an intergenic region (4q12) located between the CHIC2 and PDGFRA genes, indicating this 4;11 translocation does not result in the predicted NUP98/PDGFRA gene fusion as inferred from FISH and conventional chromosome results. This case demonstrates the clinical utility of MPseq, particularly for characterizing novel gene fusion events which may ultimately identify a false-positive FISH result.

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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    In hematologic malignancies, SVs involving enhancer hijackers can reposition promoter and/or enhancer elements of the genome (such as immunoglobulin enhancer sequences) and drive the overexpression of a nearby oncogene without producing in-frame chimeric gene fusions.53-55 These types of abnormalities are relatively common in lymphoid malignancies and have been shown to lead to false-negative56-58 or, in rare instances, false-positive FISH results.59 For example, the MYC break-apart FISH probe demonstrates an approximately 4% false-negative rate in diffuse large B-cell lymphoma56 and a 50% to 70% false-negative rate in PCNs.58,60

  • Utilizing next-generation sequencing to characterize a case of acute myeloid leukemia with t(4;12)(q12;p13) in the absence of ETV6/CHIC2 and ETV6/PDGFRA gene fusions

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    Lastly, this case demonstrates the superiority of NGS-based assays in resolving structural chromosomal abnormalities when compared to traditional cytogenetic techniques, including conventional chromosome and FISH studies. Specifically, MPseq has been shown to detect cryptic gene rearrangements, resolve false-positive FISH results, identify novel gene fusions and provide a genome-wide evaluation of recurrent genetic abnormalities observed in hematologic neoplasms [14–17]. In summary, we report a case of AML with t(4;12)(q12;p13) that could not be fully characterized by traditional cytogenetic techniques.

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