A Cryptic ETV6/ABL1 Rearrangement Represents a Unique Fluorescence In Situ Hybridization Signal Pattern in a Patient with B Acute Lymphoblastic Leukemia

Although rearrangements involving each ETV6 and ABL1 gene are some of the most commonly observed chromosomal translocations in hematological malignancies [1, 2], an ETV6/ABL1 rearrangement is very rare, and only 29 such cases have been reported to date [3]. This fusion has been associated with a wide range of hematological malignancies, including 11 cases of atypical chronic myeloid leukemia (BCR/ABL1 negative), 3 cases of myeloproliferative neoplasm (MPN), 5 cases of acute myeloid leukemia, and 10 cases of ALL. The rarity of this rearrangement is related to the inverse transcriptional orientation of the 2 genes relative to the centromeres, because it would involve at least 2 events to form an in-frame fusion transcript [3]. Here, we report a case of B-ALL with ETV6/ABL1 rearrangement.

A 30-yr-old woman, 12 weeks pregnant with her second child, visited the Samsung Medical Center emergency room because of a complete blood count (CBC) abnormality detected at a referring hospital. She experienced cough and sore throat for 3 weeks prior to her visit, and headache, dyspnea, and tender gums occurred several days before presentation. A peripheral blood count revealed white blood cell counts 227.47×10⁹/L with 85% blasts, hemoglobin 9.3 g/dL, platelet counts 39×10⁹/L, and serum lactate dehydrogenase 2,316 U/L. Bone marrow aspirate smear revealed diffuse infiltration by lymphoblasts (75%), which expressed CD19, CD10, CD20, cCD22, cCD79a, HLA-DR, CD34, terminal deoxynucleotidyltransferase (TdT), and aberrant CD33; thus, the patient was diagnosed as having B-ALL. Conventional chromosome analysis of bone marrow cells indicated trisomy 5 as the sole abnormality in 19 of 20 metaphases analyzed. Multiplex reverse transcription (RT)-PCR indicated positivity for the 1,141-bp ETV6/ABL1 type B fusion transcript (exons 1-5 of ETV6 fused to exon 2 of ABL1) and also weak positivity for the type A fusion transcript (exons 1-4 of ETV6 fused to exon 2 of ABL1). Sequence analysis of fusion transcripts using the Sanger method confirmed the fusion of ETV6 exon 5 to ABL1 exon 2, and the existence of 2 different transcripts, type A and B, was evidence for alternative splicing (Fig. 1G).

Fig. 1.

FISH analysis using locus-specific identifiers for interphase and metaphase cells of bone marrow aspirate. (A) LSI ETV6/RUNX1 (ES) probe on interphase cells, (B) LSI ETV6/RUNX1 (ES) probe on metaphase cells, (C) LSI BCR/ABL1 (DF) probe on interphase cells, (D) LSI BCR/ABL1 (DF) probe on metaphase cells, (E) LSI ETV6 (BA) probe on interphase cells, (F) LSI ETV6 (BA) probe on metaphase cells, and (G) Sequence of fusion transcript products spanning the fusion site, revealing fusion of ETV6 exon 5 to ABL1 exon 2.

Abbreviations: LSI, locus-specific identifier; DF, dual fusion; ES, extra signal; BA, break-apart.

Since HemaVision (DNA Technology, Aarhus, Denmark) and Sanger sequencing had already revealed the ETV6/ABL1 rearrangement, we performed FISH analysis using the following commercially available locus-specific identifiers (LSI): ETV6/RUNX1 Extra Signal (ES), BCR/ABL Dual Fusion (DF), and ETV6 Break Apart (BA) DNA probes (Abbott Vysis, Des Plaines, IL, USA). LSIs were used in both interphase and metaphase cells of bone marrow aspirate (Fig. 1A-F). The BCR/ABL DF probe revealed interphase cells with 2 green signals for BCR and 3 orange signals for ABL1, suggesting a rearrangement of ABL1. The ETV6/RUNX1 ES probe revealed 2 green and 2 orange signals for ETV6 and RUNX1, respectively, with a third, albeit significantly smaller, ETV6 signal. The ETV6 BA probe revealed an additional orange signal for the 5' end of ETV6 with 2 normal fusion signals. These results were different from what we had expected, since ETV6/ABL1 rearrangement with a breakpoint at intron 5 of ETV6 may not affect the ETV6 signal, encompassing exons 1-5 of ETV6 on the ETV6/RUNX1 ES probe, and also may not produce split ETV6 signals on the BA probe. Metaphase cell analysis revealed abnormal signals for BCR and ETV6 on chromosomes 12 and 9, respectively.

Collectively, these results indicate a fusion between ETV6 and ABL1 with inverse chromosomal orientation, which could occur via 2 events (Fig. 2): 1) a balanced t(9;12)(q34;p13) results in the juxtaposition of part of ABL1 (exons 2-10) in the vicinity upstream of ETV6, exon 1 at 12p13; followed by 2) an inversion within the 12p13 segment of der(12)t(9;12) after breakage at intron 5 of ETV6, and the boundary of the ABL1 segment bordered by upstream of ETV6. The International System for Human Cytogenetic Nomenclature (ISCN) for the abnormal clone demonstrated by FISH is: 47,XX,+5. ish der(9)t(9;12)(q34;p13),der(12)t(9;12)(q34;p13)inv(12)(p13p13). The patient received induction chemotherapy and, after consolidation treatment, underwent allogenic peripheral blood stem cell transplantation; however, she experienced relapse of B-ALL 5 months after transplantation.

Fig. 2.

Schematic representation of the suspected chromosomal mechanism leading to ETV6/ABL1 rearrangement in this case.

There are various descriptions in the literature of in-frame fusion products involving ETV6 and ABL1 with varying orientations, but ETV6/ABL1 rearrangement is often not detected using conventional chromosome analysis because of the cryptic nature of this rearrangement [4]. These cryptic rearrangements were also due to similar G banding patterns of the long arm of chromosome 9 and short arm of chromosome 12. RT-PCR screening for leukemia-related fusion transcripts (using methods such as HemaVision) is a useful tool to detect these cryptic rearrangements; however, this RT-PCR screening has been mainly performed in acute leukemia and not in other hematologic malignancies, such as MPN. Therefore, the frequency of ETV6/ABL1 rearrangement among total hematologic malignancies is likely underestimated.