Since the first description of a Ph negative BCR/ABL1 positive CML patient , several studies have been published reporting similar cases. Most of them are focused on the presence and location of the BCR/ABL1 fusion in CML patients with masked Ph chromosome and commonly achieved by application of commercial FISH probes, which have been proved to be very useful to identify the presence and location of the BCR/ABL1 fusion gene in CML patients with no distinguishable Ph chromosome. These studies have established the importance of FISH tests for the diagnosis and therapy monitoring of Ph negative BCR/ABL1 positive CML. However, the commercial probes don't provide enough information to understand the mechanisms involved in the formation of the masked Ph chromosome.
We used FISH mapping with BAC probes in order to study the formation of the BCR/ABL1 fusion and the underlying genomic rearrangements in 9 CML patients with Ph negative BCR/ABL1 positive CML and the cell line CML-T1. The formation of the fusion gene resulted from the relocation of not only the 3' ABL1 sequences within the BCR region at chromosome 22q11.2 or 5' BCR sequences within ABL1 region at 9q34.1, but also a considerable amount of flanking material, leaving the chromosome morphology apparently intact.
The fusion gene was located at 9q34.1 in 3 patients, at 22q11.2 in 5 patients and CML-T1, and at 22p11 in another patient. 5 out of the 6 cases with a 9q34 insertion at 22q11 displayed recurrent distal breakpoints that fell within two gene bearer regions. Thus, in 3 patients a common breakpoint cluster of 280 Kb was found. According to UCSC database, this region houses 3 genes: POMT1 (protein-O-mannosyltransferase1), UCK1 (uridine-cytidine kinase 1) and RAPGEF1 (guanine nucleotide releasing factor for RAP1; also known as C3G). In another patient and CML-T1 the breakpoint fell within a single BAC clone encompassing the 3' end of RXRA gene (retinoid × receptor alpha). Although further investigations were not carried out in this study, both RAPGEF1 and RXRA belong to pathways the disruption of which may be relevant to the evolution of the patients. RAPGEF1 has been shown to have transformation suppressor activity towards several oncogenes [20, 21] and also interact with BCR/ABL1 . RXRA belongs to a family of nuclear receptors that target multiple signalling pathways  and its downregulation has been showed to be critical for neutrophil granulocytes differentiation . Other studies found RXRA to contribute in acute promyelocytic leukaemia transformation [25, 26]. Although we have no direct evidence for the immediate involvement of either RAPGEF1 or RXRA genes, their potential role merits further investigation.
Two other mapping studies that found similar insertions have been published [7, 8]. In the first study, the authors used FISH mapping to identify the rearrangements involved in 2 Ph negative CML patients with variant translocations. A 3 Mb insertion from 22q11 into ABL1 was identified in 1 patient, while the other had a 9q34 insertion at the BCR region with a distal breakpoint falling within the clone RP11-353C22 (genome address: 31,278,002–131,588,248). This result matches with our findings since the latter BAC is located within the same 280 Kb common breakpoint region identified in 3 patients of our study. On the other hand, Valle et al.  found a 5.6 Mb insertion of 9q34 sequences into BCR. This insertion is larger than the ones identified in our study and was not accompanied by any deletions or other rearrangements.
Deletions of 5' ABL1 and/or 3' BCR sequences at the der(9) chromosome in patients with classical and variant Ph translocations  have been shown to have adverse prognostic value in CML patients treated with interferon , although their impact in patients being treated with tyrosine kinase inhibitors is controversial [29–31]. Dual colour, dual fusion translocation FISH probes spanning the BCR and ABL1 genes are very useful for revealing these events but they have a limited value in interphase nuclei in patients with masked Ph, since often the merging of the 5' BCR and 3' ABL1 signals by simple insertion leads to an apparent loss of one fusion signal that can be falsely assessed as deletion . Thus, D-FISH (Vysis) in a patient with a direct ins(22;9) gives a 2 red, 1 green, 1 fusion signal pattern, which is the same pattern obtained in case of a typical t(9;22) with deletion of 5' ABL1 at der(9). If the patient has a direct ins(9;22) the D-FISH signal pattern is 1 red, 2 green, 1 fusion, which could be mistaken for a typical t(9;22) with deletion of 3' BCR at der(9). Furthermore, Ph negative BCR/ABL1 positive patients with either a cryptic deletion of 5' ABL1 or 3' BCR show a 1 red, 1 green, 1 fusion D-FISH pattern, also typical for a t(9;22) with deletion of the reciprocal ABL1/BCR fusion. Therefore, when a deletion signal pattern is detected by interphase FISH with a dual colour, dual fusion probe, it is essential to look also at the metaphases in order to be able to differentiate a classical t(9;22) with deletion from a simple insertion.
Batista et al  reported a Ph negative BCR/ABL1 positive patient with an insertion of ABL1 into BCR associated with a deletion of the ASS – 5' ABL1 region. Zagaria et al  also reported two cases of CML patients with masked Ph and associated deletions: one patient with a cryptic ins(9;22) and a deletion of 5' ABL1 and 3' BCR regions; the other patient with a multi-step variant translocation and a deletion of around 400 Kb telomeric of the ABL1 gene. In addition to them, De Melo et al  identified with commercial triple-colour FISH probes 5' ABL1 deletions in 2 patients and 3' BCR deletion in 1 patient out of 14 CML patients with masked Ph. Our study confirmed and sized such deletions in 2 patients which where also part of De Melo's cohort. CML-T1 also had a 8.7 Mb deletion of 9q34 material in one of the sub-clones, but in this case the loss was found to affect the homologue not involved in the BCR/ABL1 formation.
We identified a duplication of the chromosome 22 harboring the BCR/ABL1 fusion accompanied by loss of the normal homologue in 1 out of 9 patients in this study plus the cell line CML-T1. Such duplications of the BCR/ABL1 bearing chromosome (either chromosome 22 or 9) seem to be a relatively common event in Ph negative BCR/ABL1 positive CML patients, being accompanied by loss of the normal homologue in most of the cases and seen both in chronic phase and blast crisis [33–35].
Regarding the formation of the fusion gene in Ph negative BCR/ABL1 positive CML patients, Morris et al  suggested two possible mechanisms: a one step model, where BCR/ABL1 results from a simple insertion of either 3' ABL1 into BCR or 5' BCR into ABL after three genomic breaks; and a multiple step model, with an initial classical t(9;22)(q34;q11) followed by a second translocation of both products and/or a third chromosome, requiring a minimum of 4 genomic breaks.
Our study provided evidence for the existence of both mechanisms implicated in the formation of the fusion gene in Ph negative patients. We found a simple insertion (one step event) in 6 out of 9 patients (no 1–3, 8–10) and CML-T1 (no 5), with no evidence of secondary rearrangements within the regions flanking BCR and ABL1 breakpoints apart from the accompanying deletions seen in 2 patients and CML-T1. On the other hand, traces of sequential rearrangements indicating a multiple step mechanism were found in 3 patients. Patient no 4 had a 9q34 insertion at chromosome 22 with bits from 22q11 distal to the breakpoint embedded within the der(9), suggesting an initial t(9;22)(q34;q11) followed by further translocation of both products. Patient no 7 carried the BCR/ABL1 fusion at 22p11. Sequences downstream of the breakpoint remained at their original location, while 5' BCR and all 9q34 sequences distal to ABL1 breakpoint (including the telomere) were relocated at 22p11, which could be explained by an initial t(9;22)(q34;q11) followed by a three-way translocation between 9q34, 22q11 and 22p11. This sequence of events would have required 5 breaks (2 for the translocation and 3 for the second one). However, an initial ins(9;22) followed by a reciprocal translocation between 9q34 and 22p11 would also require 5 breaks and therefore cannot be ruled out. Finally, patient no 6 had a three way cryptic t(9;22;16)(q34;q11;q?13) with 9q34 sequences embedded within the der(22), suggesting an initial ins(22;9) followed by a translocation between chromosomes 16 and der(22)ins(22;9). These data show not only that the two mechanisms do happen, but also that they are not excluding options. An example of the latter is patient no 6, where an initial direct ins(22;9) would be part of the spectrum of rearrangements that had restored the normal morphology of the der(22).