A novel cytogenetic abnormality t(7;8)(p11.2:q11.2) and a four-way Philadelphia translocation in an imatinib mesylate-resistant chronic myeloid leukemia patient

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome, created by a reciprocal translocation t(9:22)(q34;q11) which forms the chimeric gene, BCR-ABL. Variant Ph chromosome translocations involving chromosomes other than 9 and 22 have been identified in 5–10% of CML cases. Four-way Ph chromosome translocations are an extremely rare event in myeloid malignancies and the phenotypic consequences of such rearrangements have not been investigated. Deletions in chromosome 9 are known to be associated with a poor prognosis. In the present study, a novel case of Ph chromosome-positive CML in blast crisis is reported. A four-way Ph translocation was identified, involving five chromosomal regions, 9p21, 9q34, 12p13.3, 20q11.2 and 22q11.2, as well as an unbalanced translocation, der(7)t(7;8)(p11.2;q11.2). Since the majority of CML cases are currently treated with imatinib, variant rearrangements in general have no specific prognostic significance, although the mechanisms involved in resistance to therapy have yet to be investigated. In the present case, multiple partial deletions, including ABL and ASS genes on chromosome 9, the region 7p11.2 to 7pter, 8q11.2 to 8pter and two regions on chromosome 12, were identified. An additional Ph chromosome was also detected. Immunophenotyping indicated that the patient had biphenotypic leukemia. The patient did not respond positively to imatinib chemotherapy and died for unknown reasons, one month after diagnosis. The underlying mechanisms and prognostic implications of these cytogenetic abnormalities are discussed.


Introduction
Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the Philadelphia (Ph) chromosome. This chromosome is created by a reciprocal t (9:22) translocation which transfers the Abelson (ABL) oncogene on chromosome 9 to the breakpoint cluster region (BCR) of chromosome 22, resulting in the formation of a fused BCR/ABL gene (1). BCR/ABL produces an abnormal tyrosine kinase that causes aberrant myelopoiesis identified in CML. Variant Ph chromosome translocations involving one or more chromosome regions in addition to chromosomes 9 and 22 have been identified in 5-10% of CML patients (2). In these variants, four-way Ph chromosome translocations are rare (3). The prognostic significance of variant Ph chromosome CML remains unclear.
The progression of CML from the chronic phase (CP) to blast crisis (BC) is frequently associated with non-random secondary chromosomal aberrations, including +8, i(17q), +19 and an extra Ph chromosome (4).
Since tyrosine kinase activity is required for the transforming function of the BCR/ABL fusion protein, imatinib, a specific inhibitor of the kinase, is an effective treatment for CML patients. The 5-year estimated overall survival rate for patients who receive imatinib as initial therapy is 89%. This rate is higher than those reported in previous studies of CML treatment and only 7% of all patients progressed to the accelerated phase (AP) or BC (5). In a previous study, deletions on the derivative chromosome 9 [der (9)] were identified at a higher frequency in patients with variant Ph translocations than in those with classic Ph (45 and 17%, respectively) (6).

Materials and methods
Case report. In April 2011, a 22-year-old female presented with a white blood cell count (WBC) of 97x10 9 cells/l (neutrophils, 21; lymphocytes, 73; eosinophiles, 4; monocytes, 1; and basophiles, 1%). The platelet count was 268x10 9 cells/l and the hemoglobin level was 9.1 g/dl. Physical examination revealed splenomegaly and weight loss was noted. Chromosome analysis using banding cytogenetics demonstrated a karyotype consistent with clinical diagnosis of a CML in CP. The patient was treated daily with Zyloric (300 mg) and hydroxyurea (500 mg) for four days. LDH was 1860 U/l (normal, <460 U/l) and serum alkaline phosphase was 348 U/l (normal, <232 U/l). In September 2011, the patient presented for the second time with a WBC count of 132.4x10 9 cells/l (neutrophils, 1; lymphocytes, 40; and immature cells, 52%). Platelet count was 22x10 9 /l and the hemoglobin level was 10 g/dl. Imatinib mesylate (400 mg/day) was administered for five months and following this period the described symptoms were not observed. In October 2011, the patient died for unknown reasons under treatment.
Chromosome analysis. Chromosome analysis using GTG-banding was performed according to standard procedures (7) prior to chemotherapeutic treatment. A total of 20 metaphase cells derived from unstimulated bone marrow culture were analyzed. Karyotypes are described according to the International System for Human Cytogenetic Nomenclature (8).

Reverse transcription polymerase chain reaction (RT-PCR)
for BCR/ABL fusion transcripts. Total RNA was extracted from the diagnostic peripheral blood sample using the InviTrap RNA kit (Invitek GmbH, Berlin, Germany) according to the manufacturer's instructions. cDNA was prepared from 5µg total RNA with the Genequality BCR-ABL kit (AB Analitica, Padova, Italy) according to the manufacturer's instructions.
Flow cytometry immunophenotyping. Immunophenotyping of leukemic blasts was performed as previously described (7).
DNA sequencing. Detection of BCR/ABL mutation domain was performed using previously described primers (9).
DNA sequencing of the BCR/ABL kinase domain identified no mutations.
Four-way Ph translocation is extremely rare and only anecdotal cases have been described in the imatinib era. In the most recent study of CML, only 3/500 patients receiving imatinib mesylate as a frontline therapy were observed to have a four-way translocation (3).
The mechanism of development of this complex rearrangement may include a primary standard t(9;22), followed by a subsequent three-way translocation affecting chromosomes 12, 20 and the der(9). The fusion BCR/ABL signal was identified on der (22) and chromosome 22 had not rearranged with chromosome 20 or 12. These observations are consistent with a common two-step rearrangement process (12). Therefore, an inherent implication of the two-step mechanism is that variant translocations may be associated with a poorer prognosis (13).
Resistance to chemotherapy occurs as a result of increased expression of the BCR-ABL kinase from genomic amplification, clonal chromosomal evolution or mutations in the ABL kinase of the BCR-ABL gene, affecting drug interaction or kinase activity (14).
Submicroscopic ASS gene deletions in fused chromosome 9 were previously reported to be important for development of shortened CP and decreased overall survival, associated with a poor prognosis and response to interferon and imatinib mesylate (15,16).
Leukemias of ambiguous lineage are uncommon, representing ~4% of all acute leukemias, and frequently demonstrate an aggressive disease course, with mean survival rates less than those of leukemias derived from a single-cell lineage (17). No single chromosome abnormality is unique to biphenotypic leukemia (18). In the present study, a complex cytogenetic abnormality was identified using conventional and molecular cytogenetics. Therefore, we hypothesize that leukemias of ambiguous lineage associated with cytogenetic abnormalities indicate a poorer prognosis than those without demonstrable abnormalities.
In conclusion, the present study reports a novel case of a Ph chromosome-positive CML in BC with a four-way Ph translocation. The translocation is likely to result from a two-step mechanism. In addition to an unbalanced translocation der(7) t(7;8)(p11.2;q11.2), multiple partial chromosomal regions were deleted, partial monosomies 8, 12 and an additional Ph chromosome were identified. Immunophenotyping indicated that the patient had biphenotypic leukemia. These observations represent an adverse prognosis in CML. The patient died under treatment one month after diagnosis. Department, for their support. The present study was supported by the AECS, and in part by the DAAD, Stefan-Morsch-Stiftung and the Monika-Kutzner-Stiftung.