EP060: Characterizing the complex chromosomal rearrangements in a Philadelphia negative BCR/ABL1 positive case

Abstract

The Philadelphia chromosome (Ph), produced by the reciprocal translocation t(9;22)(q34;q11.2), is a derivative chromosome 22 with a chimeric BCR/ABL1 fusion gene encoding constitutively active tyrosine kinase protein. The Ph occurs in 95% of chronic myeloid leukemia (CML) cases and the BCR/ABL1 fusion is a diagnostic hallmark for CML. A small subset of patients (around 5-10%), displaying typical features of CML, lack a Ph by chromosome analysis, but are shown to carry BCR/ABL1 fusion either by fluorescence in situ hybridization (FISH) or molecular analysis. Two major mechanisms have been proposed to explain how BCR/ABL1 rearrangements arise in these Ph-negative BCR/ABL1 positive cases: a cryptic insertion between chromosomes 9 and 22; or via two sequential translocations where a standard t(9;22) translocation is followed by an additional reverse translocation resulting in the reconstitution of the normal morphology of the partner chromosomes. The bone marrow specimen of a 66 year old female patient, presented with persistent leukocytosis with myelocytes and metamyelocytes, along with thrombocytosis, was sent to our lab for both BCR/ABL1 RT-PCR analysis and chromosome analysis, due to suspicious myeloproliferative neoplasm. The RT-PCR analysis showed the presence of the BCR/ABL1 major (p210) breakpoint fusion transcript b2a2 (e13a2). Initial chromosome analysis did not identify a typical t(9;22)(q34;q11.2) reciprocal translocation; instead, structural abnormalities were observed on the short arm of chromosome 12 and the long arm of chromosome 22. Overall, the abnormal chromosome 22 resembles the morphology of a “Philadelphia chromosome” while both homologs of chromosome 9 show normal morphology. This leads us to hypothesize that a complex rearrangement involving chromosomes 9, 12, and 22 might be contributing to this case. Metaphase fluorescence in situ hybridization (FISH) analysis was performed to test our hypothesis using BCR/ABL1 fusion probes, where the only cryptic BCR/ABL1 fusion signal was mapped to the long arm of chromosome 9 that has normal morphology at band q34, while the long arm of the derivative chromosome 22 is absent for BCR/ABL1 fusion signals, suggesting that the Ph-like derivative chromosome 22 is not a real Philadelphia chromosome, making this case a true Ph negative case. The final ISCN for the case is designated as: 46,XX,der(9)(9pter->9q34::22q11.2->22q11.2::9q34->9qter),der(12)(22qter->22q11.2::12p13->12qter),der(22)(22pter->22q11.2::12p13->12pter), and this can be explained by an initial three way translocation between chromosomes 9, 12, and 22 followed by an additional sequential reverse translocation between the derivative chromosomes 9 and 22, leading the cryptic BCR/ABL1 fusion being inserted into the long arm of chromosome 9 and bringing chromosome 9 back to a normal morphology. Cryptic BCR/ABL1 fusion has been characterized as a variant rearrangement in CML patients lacking Ph, and up to 50% of these cases with variant BCR/ABL1 fusion exhibit a del(9q) of the derivative chromosome 9, which has been considered to be a marker for poor prognosis in CML patients. Thus, chromosomal microarray analysis (CMA) was also performed for this case, and no deletions in 9q were identified. Here, we presented a case where FISH mapping was utilized to further characterize the complex chromosomal rearrangements between chromosomes 9, 12, and 22 in a Ph negative BCR/ABL1 positive case, which reveals that the cryptic BCR/ABL1 is inserted into the long arm of chromosome 9 with normal morphology at band q34, while the Ph-like derivative chromosome 22 is not a true Ph chromosome. Our study illustrates the challenge of recognizing the complex pattern of cytogenetic aberrations in these Ph negative BCR/ABL1 positive CML cases with variant BCR/ABL1 rearrangements, and highlights the importance of utilizing comprehensive cytogenomic and molecular techniques (including metaphase FISH, RT-PCR and CMA) in characterizing these complex chromosomal abnormalities for proper diagnosis and prognosis prediction.