Complex Variant t(9;22) Chromosome Translocations in Five Cases of Chronic Myeloid Leukemia

The Philadelphia (Ph1) chromosome arising from the reciprocal t(9;22) translocation is found in more than 90% of chronic myeloid leukemia (CML) patients and results in the formation of the chimeric fusion gene BCR-ABL. However, a small proportion of patients with CML have simple or complex variants of this translocation, involving various breakpoints in addition to 9q34 and 22q11. We report five CML cases carrying variant Ph translocations involving both chromosomes 9 and 22 as well as chromosomes 3, 5, 7, 8, or 10. G-banding showed a reciprocal three-way translocation involving 3q21, 5q31, 7q32, 8q24, and 10q22 bands. BCR-ABL fusion signal on der(22) was found in all of the cases by FISH.


Introduction
Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph 1 ), resulting from a balanced translocation between the long arms of chromosome 9 and 22, the t(9;22)(q34;q11.2) [1]. In the formation of the Ph 1 chromosome, the 3 region of the c-ABL oncogene is transposed from 9q34 to the 5 region of the BCR gene on chromosome 22 to form a fusion gene BCR-ABL, which encodes a fusion protein with constitutive tyrosine kinase activity [2]. Although the vast majority of patients with CML show the classical t(9;22)(q34;q11.2) translocation, variant Ph translocations are present in 5%-10% of CML cases. These are cytogenetically classified as simple variants involving chromosome 22 and a chromosome other than 9, and complex variants that involve chromosomes 9, 22, and one or more other chromosomes [3]. In almost all the cases with variant Ph 1 chromosome, the BCR-ABL rearrangement can be detected by molecular methods or by fluorescence in situ hybridization (FISH).
In this work, we described five patients diagnosed with CML carrying different complex variant Ph translocations involving chromosomes 9, 22 as well as one other chromosome. They were studied by G-banding, FISH, and reverse transcription-polymerase chain reaction (RT-PCR). . For detecting BCR/ABL fusion transcript, the samples were analyzed using the primers and specific labeled probes described by Bolufer et al. [5]. BCR/ABL amplified products were normalized to ABL amplifications for each sample using the primers A2 and CA3 described by Cross et al. [6].

Results
The group of five patients consisted of 3 females and 2 males, ranging in age at diagnosis from 50 to 75 years. All the patients were in chronic phase at presentation and were treated accordingly to what was considered the standard treatment in each moment receiving hydroxyurea, interferon-α and imatinib. One patient underwent autologous peripheral blood stem cell transplantation after failure of interferon-α. Cytogenetic analysis by G-banding revealed the presence of five reciprocal three-way variant translocations of the classical t(9;22)(q34;q11). The chromosome breakpoints involved in these complex variant translocations were the following: 3q21, 5q31, 7q32, 10q22 and 8q24 (Figure 1). In addition, patients (a) and (d) also present additional abnormalities: patient (a) showed a complex karyotype with at least two main unrelated clones, whereas patient (d) showed numerical abnormalities of chromosomes 15 and 22. BCR/ABL dual-color FISH demonstrated in all cases  Table 2. At present, all the patients are doing well in hematological and complete cytogenetic remission following standard dose imatinib treatment, except for patient (a) who died of congestive heart failure not related with imatinib treatment. Patients (d) and (e) showed complete disappearance of

Discussion
In the present report we analyzed five patients with CML carrying complex Ph 1 translocations involving various partner chromosomes by cytogenetics, FISH, and molecular methods. In each case, chromosomal translocations lead to a BCR-ABL fusion, as occurs in the standard t(9;22) translocation [2]. The third chromosome present in each of these variant translocations is known to be implicated in some cases of Ph-positive CML cases. Besides, the involvement of bands 3q21 (3 cases), 5q31 (2 cases), 7q32 (1 case), 8q24 (3 cases), and 10q22 (9 cases) had also been previously reported in other cases of CML [7]. Nevertheless, it becomes difficult to report the exact number of cases with such complex translocations due to the large amount of variability in cytogenetics nomenclature observed before ISCN, 2005. Evaluation of the prognostic significance of these translocations has been analyzed in case reports or small series giving controversial results. However, it has been recently reported that patients with variant translocations have a similar prognosis to those with classical Ph 1 translocations when treated with imatinib mesylate [8][9][10]. In our series, all the patients are at present in hematological and complete cytogenetic remission following standard-dose imatinib treatment after 12 to 86 months of follow-up. Regarding molecular remission, patients (d) and (e) showed complete disappearance of the fusion transcript after 71 and 86 months, respectively. Patients (a)-(c) did not reach complete disappearance of the fusion transcript but reduced the levels in more than 2 logs. Patient (a) showed a lighter reduction probably due to the complex karyotype at diagnosis.
Deletions of der (9) have been recognized in 10%-15% of patients in the chronic phase, being more frequently found in variant translocations. These deletions are thought to occur at the time of the Ph 1 translocation and are known to be associated with a worse survival [11]. However, a recent study has suggested that imatinib mesylate may overcome the adverse prognostic significance of der(9) deletions [12]. In our study, none of the patients had a deletion of a sizable portion on the derivative chromosome 9.
In conclusion, we described five low-frequency complex variant t(9;22) translocations representing 6% of the CML cases diagnosed in our center during approximately sevenyear period. Despite low numbers, in our experience patients carrying complex Ph 1 translocations do not differ significantly in hematological and clinical features from those with standard translocation.