The Philadelphia (Ph) chromosome is a common cytogenetic abnormality in hematologic malignancies. Although it is most frequently associated with Chronic Myelogeneous Leukemia (CML), it can also be present in Adult Precursor B-Lymphoblastic Leukemia/ Lymphoma (ALL) and less commonly in pediatric and Adult Acute Myelogeneous Leukemia (AML) [1]. At the molecular level, the t (9; 22) (q34; q11) juxtaposes the 5’ end of the breakpoint cluster region (BCR) gene on chromosome 22 to the 3’ end of the Abelson Tyrosine Kinase (ABL) gene on chromosome 9. CML in blast crisis is often accompanied by the presence of additional chromosomal aberrations [1]. Amongst those, activation of the EVI1 (Ectopic Viral Integration Site 1) gene has been reported in a small percentage of patients; ectopic expression of the EVI1 gene is usually due to recurrent 3q26 rearrangements such as the t(3;21)(q26;q22) and the inv(3;3)(q21q26) [1]. The t (3;21) (q26;q22) resulting in the AML1/MDS1/EVI1 (AME) fusion is reported in t-AML, t-MDS (i.e. myelodysplastic syndrome) and in CML [2]. In this translocation parts of the AML1 gene could be fused to one of the two aforementioned genes which are adjacent in 3q26. These fusion products, in cooperation with other genetic abnormalities, are capable of blocking myeloid differentiation possibly by interfering with the normal transcriptional regulatory functions of AML1. Furthermore, they initiate malignant transformation in cell lines and myeloid leukemia in mouse tumor models. However, the clinical features of t(3;21)-associated human leukemia have not been well defined to date due to the lack of any large clinical case studies [3].

The association of acquired trisomies involving various chromosomes and hematological malignancies is well established. The presence of some single acquired autosomal trisomies may be indicative for prognosis, too [4]. Trisomies 9 and 10 are rare observed in AML [4,5].

Here we reported a Ph chromosome positive Acute Myelogeneous Leukemia Subtype M5 (AML-M5) which transformed from a Chronic Myelogeneous Leukemia (CML) presenting multiple trisomies, including a double t (9; 22) (q34; q11) and an AME fusion transcript resulting from a t(3;21)(q26;q22).

A 27-year old male was diagnosed with CML in chronic phase in March 2010 due to splenomegaly. In June 2011, the patient presented for the third time (Table 1) with a WBC of 4.3×10⁹/l consisting of 48.6% neutrophils, 27.7% lymphocytes and 23.7% monocytes. The platelets count was 55×10⁹/l and the hemoglobin level was 10.1 g/dl. The serum LDH level was 1024 U/l and the serum Alkaline Phosphatase (ALP) level was 301 U/l (normal level up to 300 U/l). The patient was treated with Imatinib mesylate (IM) at 400 mg/day for overall 15 months in the total; treatment regime was not altered as still the clinicians expected to have a positive effect from it even after blastic change was evident. In October 2011, the patient presented for the fourth time, having a WBC of 4.4×10⁹/l consisting of 63% neutrophils, 28.4% lymphocytes and 8.4% monocytes (Table 1). The platelets count was 70×10⁹/l and the hemoglobin level was 12 g/dl. The serum LDH level was 900 U/l. The patient was treated with Nilotinib at 800 mg/day for overall 4 months. In November 2011, he passed away under the treatment due to unknown reasons.

Table 1: Clinical data of the patient together with the corresponding cytogenetic findings.

Karyotyping was performed one time before, two times during IM treatment and one time under Nilotinib treatment (Table 1). Initially a CML-specific karyotype 46, XY, t (9; 22) (20) was found, which evolved within 6 months under IM therapy to a more complex one: 46, XY, t (3; 21) (q26; q22), t (9; 22) (q34; q11.2) (7) /46, XY (13). This evolution continued and 15 months after diagnosis the karyotype was 51, XY, t (3;21) (q26;q22), +8, +9, t (9;22) (q34;q11.2) x 2, +10, +12, +22 (20) (Figure 1 and 2). Interestingly, under Nilotinib the karyotype became less complex and was: 49, XY, t (3; 21) (q26; q22), +8, t (9; 22) (q34; q11.2) x2, +10, +22 (16) /46, XY [4].

Figure 1: In June 2011 GTG-banding revealed trisomies 8, 9, 10, 12 and 22 as well as rearrangements of chromosomes 3 and 21 in the patient (Table 1). All derivative chromosomes are marked by highlighted by arrow heads.

Figure 2: (A) Metaphase FISH using probes for BCR (green) and ABL (orange) showed four BCR/ABL1 fusion signals (yellow): two signals on the two der(9) chromosomes and two signals on the two der(22) chromosomes. The green and orange signals indicate, the BCR gene on the normal chromosome 22 and the ABL gene on the normal chromosome 9, respectively. (B) Metaphase FISH using probes for WCP21 (green) and WCP3 (red) confirmed an involvement of chromosome 3 with chromosome 21 in this case. Abbreviations: # = chromosome; der = derivative chromosome; Ph = Philadelphia-chromosome.

RT-PCR-analysis for BCR/ABL and AME fusion transcripts were done on samples taken 6, 15 and 19 months after diagnosis. Test for BCR/ABL demonstrated the presence of a p210, b2a2 fusion transcript most often found in CML at all three times (Figure 3A). DNA sequencing for BCR/ABL kinase domain did not show any mutation (data not showed). The t (3; 21) translocation was characterized in more detail by multiplex RT-PCR, which revealed a typical AME fusion transcript product of 832 bp (Figure 3B) in all three samples.

Figure 3: (A) Gel electrophoresis of the nested RT-PCR products from the patient. Lanes 1 and 3, negative control; lane 2, internal control (b3a2, 353 bp) and line 4, BCR/ABL fusion transcripts (b2a2, 278 bp), (B) Gel electrophoresis of multiplex RT-PCR products from the present case. Lane 1, negative control and lane 2, AML1-MDS1-EVI1 fusion transcript (832 bp), (C) Sequencing analysis from a case of CML with t(3;21) confirmed that the fusion transcript consists of portions of AML1, MDS1, and EVI1.

Sanger DNA sequencing confirmed the presence of AME fusion transcripts with breakpoints in MDS1 at amino acid position 115 (asparagine) and 227 (glutamic acid), and in EVI1 at amino acid 26 (methionine); the AML1 breakpoint was located at amino acid 821 (glutamine) (Figure 3C).

Immunophenotypic analysis of peripheral blood at all four times of cytogenetic studies is summarized in table 1. The immunophenotype of the last two patient samples were consistent with AML-M5 according to FAB classification.

According to the literature, a t (3; 21) (q26; q22) with multiple trisomy is a recurrent cytogenetic abnormality in two similar cases [3,6]. To the best of our knowledge, a karyotype and karyotypic evolution such as seen in the present case with expression of the AME fusion protein has never been described before in AML or CML [7]. Overall, the patient unexpectedly developed an AML-M5 from an initial CML, a development which we could also depict in terms of accompanying (cyto-) genetic changes.

It has been previously demonstrated that in t (3;21)-associated leukemia [2] and the fusion of AML1 with MDS1 may interfere with normal AML1 function and promote transformation [3]. AME may antagonize native AML1 function by interactions with other core binding complex elements including HDAC and CtBP11; its action may be modulated by acetyltransferases. A recent study suggests that AME also may act to down-regulate another core binding factor (CEBPA) by inhibition of protein translation [3]. Intergenic splicing of MDS1 and EVI1 produces a protein with important effects in normal hematopoiesis and leukemogenesis, and the AME function may also be related to inhibition of the normal role of MDS1-EVI1 in myelopoiesis [3].

In IM therapy, BCR-ABL gene amplification resulting from additional Ph copies has been identified as one of the major mechanisms of drug resistance [8]. Despite the absence of mutations in the drug-binding site, the presence of multiple copies of the BCRABL oncogene is indicative of a poor prognosis and higher possibilities for resistance to drug treatment [9]. As under IM therapy the initial one Ph chromosome duplicated, this could also have happened in the present case.

The occurrence of t (3; 21) translocation together with amplification of BCR-ABL marks the aggressiveness of disease progression [2]. Animal data demonstrate that bone marrow cells co-transfected with BCR-ABL1 and AME rapidly induce an AML-like malignancy in mice. Therefore, coexistence of an EVI1 translocation and BCR-ABL1 is sufficient to cause evolution of myeloid blast crisis [10]. Murine studies demonstrated that AME cooperated more effectively with BCR-ABL1 than MDS1-EVI1 or EVI1 in causing AML with short latency [11].

Trisomies have an overall adverse prognostic value in hematological malignancies. Trisomy 9 is rarely reported in AML, when present has intermediate prognosis; it has also been considered as marker for benzene-related leukemogenesis, common in systemic mastocytosis and myeloproliferative disorders [4]. Trisomy 10, as a sole cytogenetic abnormality, has rarely been seen in ALL and MDS. However, it is even less frequent in AML, and has an unclear prognostic impact [5].

In conclusion, here we reported a novel cytogenetically case of a Ph chromosome positive CML evolving to an AML-M5 having multiple trisomies, a double t (9; 22) (q34; q11) and AME fusion transcript resulting of t (3; 21) (q26; q22). The reported patient did not respond positively to chemotherapy. These finding are indicative that CML patients acquiring a t (3; 21) (q26; q22) might be subject to bone marrow transplantation rather than to IM-therapy.

We thank Prof. I. Othman, the Director General of Atomic Energy Commission of SYRIA (AECS) and Dr. N. Mirali, Head of Molecular Biology and Biotechnology Department for their support.

This work was supported by the AECS and in parts by the DAAD, Stefan- Morsch-Stiftung and the Monika-Kutzner-Stiftung.

AW performed the cytogenetic studies in the present case and collected the data relative to this case report; WA supervised the cytogenetic analysis; AW, MO, TL did the molecular cytogenetic analysis and interpretation; FM did the molecular biology analysis and interpretation; IA did the flow cytometry analysis and AW and TL drafted the paper and all authors contributed to the finalizing of the manuscript.