Secondary Philadelphia chromosome after non-myeloablative peripheral blood stem cell transplantation for a myelodysplastic syndrome in transformation

A secondary Philadelphia chromosome (Ph) appearing during the late course of hematopoietic malignancies is exceptional,1,2 frequently therapy-related and associated with poor prognosis.3 In addition, Ph is very rarely observed in myelodysplastic syndromes, and is usually associated with a minor breakpoint (p190Bcr-Abl).4 Here, we report the first case of a secondary Ph with a minor breakpoint at relapse after a sibling-donor nonmyeloablative peripheral blood stem cell transplantation performed for a myelodysplastic syndrome in partial remission. A 48-year-old female was referred to our department in June 2001 for pancytopenia: WBC: 1.3 109/l (neutrophils 72%, lymphocytes 25%, monocytes 2 and 1% of blasts), Hb: 79 g/l, Plt: 29 109/l. Bone marrow aspirate showed 15% of blasts and dysplastic characteristics (hypogranular neutrophil precursors, neutrophil Pelger–Huët hypolobulations, dysmegakaryocytic and dyserythropoietic features). Immunophenotyping analysis detected a CD13+ MPO+ CD33+ CD34- myeloid clone. Cytogenetic analysis on the marrow leukemic cells showed a trisomy 8 in 12 out of the 20 cells examined (Figure 1), with no structural abnormalities. The diagnosis of refractory anemia with excess of blasts in transformation (RAEB type II) was retained. The patient underwent two courses of standard AML-like chemotherapy consisting of daunorubicin and cytarabine, with a complete cytological and karyotypic remission. After 5 months, she relapsed with strictly similar clinical, cytological (41% of bone marrow blasts), immunophenotypical and cytogenetical characteristics. Since conventional chemotherapy had failed in controlling the disease, and because a full HLA-matched sister was available, in March 2002, the patient received a non-myeloablative allogeneic transplant with her sister's G-CSF-mobilized peripheral blood stem cells, as immunotherapy. Conditioning regimen consisted in 3 days of fludarabine, 2 Gy total body irradiation and cyclosporin+mycophenolate mofetil as immunosuppressive agents.5 Molecular chimerism (VNTR analysis) analyzed at day 28 after transplantation was the 93% donor. She developed at day 60 a grade II skin acute GVHD resolved after a short course of steroids. At 3 months after transplantation, the patient relapsed with WBC 10.1 109/l (neutrophils 58%, eosinophils 1%, basophils 2%, monocytes 14%, myelocytes 1%, lymphocytes 9%, blasts 15%) and disseminated intravascular coagulation. The bone marrow aspirate showed 40% polymorphic blasts with a different morphology than that from diagnosis. Megakaryocytic cells showed dysplastic features and were predominantly micromegakaryocytes. Immunophenotyping analysis showed a similar CD13+ MPO+ CD33+ phenotype, but the acquisition of CD34 expression. The karyotype showed in all 20 analyzed metaphases a Ph resulting from t(9;22)(q34;q11) associated with 5q deletion and other complex rearrangements (Figure 2), but there were no metaphases with trisomy 8 or the normal karyotype. FISH analysis using a probe for detection of the Bcr-Abl translocation (LSI BCR/ABL ES Dual Color Translocation Probe, Vysis, Woodcreek, IL, USA) exhibited two fused orange/green signals (5'Bcr/3'Abl) located on the long arms of chromosomes 9 and 22, suggesting a minor breakpoint signal pattern (Figure 3). Reverse-transcription and 'real-time' quantitative polymerase chain reaction (RQ-PCR)6 confirmed this assumption with a p190Bcr-Abl transcript detected, and no p210Bcr-Abl detectable. Molecular bone marrow chimerism analysis was 85% recipient, confirming the autologous reconstitution and excluding a secondary leukemia from donor cells. Retrospective FISH analysis performed on frozen bone marrow cells from diagnosis, showed the absence of the Bcr-Abl fusion gene and of 5q31 deletion (EGR1, LSI EGR1/D5S721, D5S23 Dual Color Probe, Vysis) among all analyzed metaphases and interphase nuclei. In addition, no Bcr-Abl transcript could be detected either on the patient's cells from diagnosis, or in G-CSF mobilized donor peripheral blood stem cells, with sensitivity thresholds of 1/103 and 1/105, respectively. Immunosuppressive therapy was withdrawn and the patient received imatinib mesylate along with chemotherapy, with partial and transient responses. She finally died from cerebral hemorrhage at day 185 after transplantation. We describe here the first case of a secondary minor Bcr-Abl Ph occurring after non-myeloablative sibling-donor allogeneic hematopoietic stem cell transplantation, with poor prognosis. Secondary Ph occurring after conventional chemotherapy or autologous hematopoietic transplantations has already been reported a few times in the literature.1,3,4 In myelodysplastic syndromes, the Ph is an unusual finding (19 cases reported to date4,7,8), and mostly concomitant with acute transformation of the disease, as in our case. Since we could perform a retrospective analysis by FISH and RTRQ-PCR on the patient and donor's frozen cells along the course of the medical history, we could ensure that (1) the Ph has really been acquired after allogeneic transplantation and was not present at diagnosis, (2) this chromosomal abnormality was developed from recipient cells and not donor cells, and this was particularly important to point out as normal donors can harbor the Bcr-Abl+ translocation,10,11 (3) the Ph acquisition occurred shortly (3 months) after allogeneic transplantation and it is probably unlikely that conventional chemotherapy given initially (started 9 months earlier) could induce such genetic lesions, as it usually takes much longer time to occur,9 (4) a different cytological (polymorphic blasts, megakaryocytic dysplastic features), immunophenotypical (CD34negative), cytogenetical (Ph and 5q deletion) and molecular (p190Bcr-Abl transcript) phenotype than that was found from diagnosis. All these last features can be encountered in myelodysplastic syndromes,4,7 which illustrates here once again the genetic instability that characterizes myelodysplastic syndromes associated with the outgrowth of a new malignant Bcr-Abl+ leukemic subclone enhanced by post transplant immunosuppression. Unfortunately, imatinib mesylate, after an initial response, subsequently failed in long-term control of the disease as in blastic-phase CMLs.12 In conclusion, this case report illustrates once again the poor prognosis of diseases with secondary Ph+ clones, and that they can be observed after non-myeloablative allogeneic stem cell transplantations, where the context of immunosuppression can certainly favour the Bcr-Abl+ clone emergence and overgrowth. This work was partially supported by a grant from the 'Ligue Contre le Cancer de la Drôme' (Valence, France).