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Characterization of Ph-negative abnormal clones emerging during imatinib therapy
Article first published online: 14 MAY 2007
Copyright © 2007 American Cancer Society
Volume 109, Issue 12, pages 2466–2472, 15 June 2007
How to Cite
Abruzzese, E., Gozzetti, A., Galimberti, S., Trawinska, M. M., Caravita, T., Siniscalchi, A., Cervetti, G., Mauriello, A., Coletta, A. M. and De Fabritiis, P. (2007), Characterization of Ph-negative abnormal clones emerging during imatinib therapy. Cancer, 109: 2466–2472. doi: 10.1002/cncr.22699
- Issue published online: 4 JUN 2007
- Article first published online: 14 MAY 2007
- Manuscript Accepted: 1 FEB 2007
- Manuscript Revised: 30 JAN 2007
- Manuscript Received: 3 JAN 2007
- Gaston County Cancer Society
- chronic myeloid leukemia;
- Ph-negative clones;
- chromosomal abnormalities;
Imatinib is a tyrosine kinase-specific inhibitor widely used for the treatment of chronic myeloid leukemia (CML). Studies reported the occurrence of additional cytogenetic abnormalities in the Philadelphia chromosome (Ph)-negative cell population emerging after treatment-induced suppression of the Ph-positive clone. These abnormalities were described in a relatively high proportion of patients treated with imatinib compared with the anecdotal reports of similar cases in patients treated with other drugs. However, the origin of these abnormalities as well as their biological and clinical significance are unknown.
The study involved 13 cases of patients diagnosed with CML carrying cytogenetic abnormalities in their Ph-negative cell population after imatinib treatment. The presence of the markers within the CD34+ stem cell compartment and the cell culture growth were analyzed and patients were followed over time.
CD34+ cells express the cytogenetic markers present in Ph− cells, suggesting a possible involvement of the stem cell population. Cultured cells showed normal growth in all but 1 patient. No growth advantage was demonstrated for the Ph-negative or the Ph-positive clone after cell culture.
After follow-up of up to 49 months, none of the patients had evolved to myelodysplasia or acute leukemia. Hypothesis regarding the biological and clinical significance of these abnormalities are formulated. Cancer 2007. © 2007 American Cancer Society.
The BCR-ABL fusion gene, and the recombinant protein deriving from the reciprocal translocation between chromosomes 9 and 22, is responsible for the myeloid cell expansion in chronic myeloid leukemia (CML).1, 2 Targeting the tyrosine kinase activity of BCR-ABL to block the pathogenetic mechanism of the disease is an appealing therapeutic strategy, which became reality with the recent introduction of imatinib (Glivec; Novartis, East Hanover, NJ). Imatinib mesylate is a 2-phenaminopyrimidine derivative that specifically inhibits the abl tyrosine kinase blocking the proliferation of CML colony-forming units granulocyte macrophage (CFU-GM) and suppressing proliferation of cell lines expressing p210 BCR-ABL.3, 4
This drug has demonstrated superior activity and high tolerability compared with other treatments for CML.5, 6 Sustained complete hematologic responses (CHR) and major and complete cytogenetic responses (CCR) are common. However, Philadelphia chromosome (Ph)-negative clones carrying cytogenetic alterations similar to those detected in patients with myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), have recently been reported in patients treated with imatinib.7, 8
We report our experience in 13 patients in chronic phase CML who developed cytogenetic abnormalities in Ph-negative cells while receiving imatinib. To acquire insight into the origin of the Ph-negative clone as well as the clinical evolution of the coexisting Ph− and Ph+ cell populations, we analyzed bone marrow (BM) cell segregation, cell culture, angiogenesis, and followed the patients for up to 4 years.
MATERIALS AND METHODS
Since April 2000 we have treated 122 CML patients in different phases of the disease with imatinib. In 13 patients the emergence of a cytogenetic abnormal clone in Ph-negative cells was seen after a median of 17.6 months (range, 5–39 months) since beginning imatinib. Ten patients began imatinib therapy in chronic phase for interferon intolerance, whereas 3 patients were treated with imatinib at diagnosis. The median age was 54.7 years, the female:male ratio was 7:6, and the median time from CML diagnosis was 42 months. None of the patients had ever progressed to accelerated or blastic phase before or after imatinib treatment. Previous treatments included hydroxyurea (HU), 6-mercaptopurine (PU), busulfan (BU), Ara-C (AC), and interferon (IFN). Two patients underwent stem cell harvest after mini-ICE therapy and 1 patient had received an autologous BM transplant.
Karyotypes at CML onset and after starting imatinib were characterized in all patients by the presence of the Ph chromosome. No additional abnormalities were detected except for a dup(1q)(q11q21) in 1 patient. All patients responded to imatinib obtaining CHR, with 3 minor, 3 major, and 7 CCR when additional abnormalities were noticed in Ph-negative cells. After patients gave their informed consent, samples for further analyses were collected.
Cytogenetic and Molecular Analyses
Complete karyotyping was performed on all patients at diagnosis, repeated at 3-month to 12-month intervals, with a minimum of 20 metaphases analyzed. Standard overnight or 24-hour to 72-hour unstimulated BM cultures were used and the results reported according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). Slides for fluorescence in situ hybridization (FISH) analysis were prepared following the manufacturer's protocols. Interphase FISH analysis was performed on 200–500 nuclei. A conservative threshold of 7% (±2 standard deviations [SD]) was considered for interphase FISH results.
Cell Separation/Cell Culture
Mononucleated BM cells were obtained by Ficoll-Hypaque density gradient centrifugation and separated into CD34+ and CD34-negative subsets using an immunomagnetic cell sorting device (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of CD34+ cells ranged from 92% to 98% in all samples as determined by flow cytometry.
Progenitor cell cultures were performed using a complete pretested mixture of methylcellulose, fetal bovine serum, bovine serum albumin, rh-SCF, rh-IL3, rh-GM-CSF, and rh-erythropoietin in Iscove medium (methocult H4434, Stem Cell Technologies, Vancouver, BC, Canada). Mononucleated cells and CD34+ enriched cells were analyzed for colony-forming capacity by plating in duplicate in 35-mm Petri dishes at 2 different cell concentrations (5–10 × 104/mL and 0.3–1 × 104). Dishes were incubated at 37°C in a humidified atmosphere of 5% CO2 in air and scored after 14 days for the presence of colonies (CFU-GM, BFU-E, and CFU-MIXED).
Peripheral Blood and BM Analysis
Complete blood counts (CBC) and peripheral blood (PB) smears on these 13 patients were carefully evaluated, with particular regard to cell morphology, white blood cell differential, presence of myeloid precursors, and blasts. BM aspirates and biopsies were evaluated for cellularity, trilineage maturation, dysplastic changes, blast percentage, and vessel area extension for angiogenesis, the latter using CD34 monoclonal antibody employed according to the avidin-biotin-peroxidase complex (ABC) methods with diaminobenzidine (DAB) as final chromogen and hematoxylin as counterstain (×4).
Cytogenetic FISH Analyses
Conventional cytogenetic/FISH data at onset and follow-up are presented in Tables 1 and 2. At the Ph-negative clone presentation, 7 patients presented with +8 in 20% to 100% cells, 1 patient presented with −7 in 10% cells, and in another patient del(7)(q31) was present in 30% of cells; −Y accounted for 20%, 10% cells with t(6;7)(p24;q21) were evidenced in 1 patient, t(2;6)(p25;q23) was found in 70% cells, whereas dup(1q) was present in all metaphases in 1 patient.
|Patient||Age, y||Karyotype||Prior therapy||Disease duration, mo||Duration of imatinib therapy, mo|
|12||68||46,XY; 45,X0,−Y ||HU||66||6|
|Patient||Follow-up, mo||% cells expressing Ph+ and Ph- clones at Δt from close evidence|
FISH analysis (bcr-abl [Cancer Genetics, River Vale, NJ]; LSI-EGFR-CEP7, LSIc-myc, and CEPX/Y [Vysis, Downers Grove, Ill]) confirmed the presence of the additional abnormalities that were noticed only on Ph-negative cells (Fig. 1B). Ph-negative abnormal cells showed the presence of 2 red signals on chromosomes 9 and 2 green signals on chromosomes 22, thus excluding the possibility of deletion of Ph as a secondary clonal evolution of CML Ph+ cells (Fig. 1A).
Two to 4 previous samples per patient, and also 1 sample pre-imatinib from archived material, were retrospectively analyzed using FISH in patients presenting +8, −7, del(7), or −Y, revealing no hidden abnormalities.
Cytogenetics and FISH were repeated in 12 patients and the abnormalities confirmed, whereas Patient 3 had a matched donor and was transplanted. Patients who lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated with the abnormal clone. The patient that became 100% Ph+ cleared the BM from the +8 clone. Patient 13 showed the emergence of the del(7) clone while receiving imatinib; this additional abnormality disappeared when he lost the partial cytogenetic remission. The patient was then given dasatinib (BMS354825, Bristol Myers Squibb, Princeton, NJ), a second-generation tyrosine kinase inhibitor, and, after 3 months of treatment, at the time of reduction of Ph+ clone, the del(7) abnormality reappeared, being still present, after 12 months treatment and a CCR. In addition, Patient 2 was treated with dasatinib after failing imatinib, achieving partial cytogenetic response, but in this case the +8 clone was not further evidenced. The patient with dup(1q) maintained 100% duplicated metaphases while clearing the BM of Ph-positive cells (constitutional karyotype was normal), suggesting the emergence of the Ph+ leukemic clone out of a preexisting clonal background. Furthermore, patients expressing −Y as an abnormality presented with a normal constitutional karyotype, thus excluding the common loss evidenced in some elderly males.
In 4 patients the abnormal clone was not evidenced in all the subsequent controls, even after a long time interval (up to 40 months), suggesting the possibility that the abnormalities could be temporary.
Of 6 patients, 5 showed a normal cultured cell growth; only 1 (Patient 6) had an abnormal growth pattern as demonstrated with reduced CFU formation affecting BFU-Es, CFU-GM, and CFU-MIXED, and colony size microclusters on both mononucleated and CD34+ cell culture (Table 3).
FISH analyses on separated CD34+ and CD34-negative cells showed that the abnormal clone was present in the CD34+ compartment, suggesting the involvement of stem cells at this level (Table 4). FISH on cultured cells did not demonstrate a growth advantage for Ph+ cells or for the new clone (data not shown).
|Patient||BM||Ph+ clone (%)||BM||Ph− clone (%)|
Peripheral Blood/Bone Marrow Morphology
CBC and PB findings were within the normal range. BM presented with reduced cellularity, normal differential, and mild dysplastic signs as seen in patients under imatinib. No significant morphologic changes were observed during the follow-up. BM biopsies in 3 patients showed mild megakaryocytic dysplasia and reduced cellularity. No fibrosis or increased angiogenesis (Fig. 2) were observed.
The introduction of imatinib has represented a major advance leading to substantial clinical and quality of life improvement in CML patients. However, several articles have drawn attention to the occurrence of clonal karyotypic abnormalities in Ph-negative cells after imatinib-induced cytogenetic response,9–11 although the origin of these abnormalities as well as their clinical significance are unclear. Earlier reports12 have suggested the possibility that residual Ph-negative stem cells damaged by previous treatments may emerge when the Ph+ clone's proliferative advantage has been abrogated by imatinib. Recently, Terre et al.13 reported a larger series of 34 patients in which retrospective FISH analysis was performed in 15 patients showing the presence of the abnormalities in pre-imatinib samples in 4 of 15 patients. In our series, all patients were analyzed in multiple previous BM samples by FISH, using a conservative threshold of 7% to avoid the risk of false-positive results. The absence of cytogenetic abnormalities and the presence in the current series of 3 previously untreated patients suggests a different mechanism.14, 15
To our knowledge, few cases of cytogenetic clonal abnormalities in Ph-negative cells after interferon treatment and development of Ph− leukemias after CML have been described,16–18 as well as cases in which Ph− and Ph+ myeloproliferative disorders coexist.19 An alternative hypothesis to explain these findings could be that the BCR-ABL fusion may not represent the initial event in the pathogenesis of CML. This multistep hypothesis implies that the Ph chromosome is a late event that determines the clinical onset of overt CML and controls the evolution of the illness.20 Through the reduction/elimination of the Ph+ clone, the ‘very first’ aberrant stem cell can expand in the BM and eventually acquire new cytogenetic abnormalities to, potentially, escape the control of the drug. In our study it appears that the percentage of the Ph-negative aberrant cells is inversely proportional to the Ph+ clone, suggesting the presence of 2 distinct cell populations in which the Ph+ clone most likely retains the proliferative advantage.
What is the clinical meaning of these cytogenetic alterations? At the time of last follow-up, the majority of our patients had been followed for more than 3 years (median follow-up, 34.6 months) and in none had their disease evolved to MDS or acute leukemia. Patients developing secondary MDS-AML, instead, once cytogenetic abnormalities are evidenced, usually rapidly develop frank MDS-AML symptoms21; the few cases described that evolved in MDS22–24 are thus probably related to a non-CML stem cell damage due to previous treatments.
Furthermore, other biological features of MDS have been studied in our patients (eg, cell culture growth, BM morphology), with particular regard to the presence and distribution of blast cells, and angiogenesis modification to reveal preclinical dysplastic manifestations.
Patients with MDS usually present with reduced growth of colonies/clusters or excessive growth (CFU-GM, CFU-MIXED, BFU-E), and the latter has resulted in an independent predictive value for leukemic evolution.25, 26 In addition, a dose-dependent growth inhibition of normal CD34+ progenitors tested in vivo after imatinib exposure has been reported.27
In the currentr series, BM morphology and cell culture studies disclosed apparently normal findings, except for 1 patient who showed abnormal colony formation.28 However, after 48 months of follow-up this patient was still receiving imatinib, with a normal CBC and differential, histologically normal BM, and a recently achieved CCR.
Increased BM angiogenesis has been assessed in different hematologic malignancies including CML and MDS through analysis of BM microvessel density (MVD).29, 30 None of our patients presented with increased MVD as estimated by CD34 immunohistochemical expression in BM biopsies, confirming the clinical feature of hematologic remission from CML and the absence of clinical and morphologic signs of MDS.
To our knowledge the current study is one of the largest series reported of patients with CML and clonal Ph-negative cytogenetic abnormalities followed over time.31 The 10% incidence of additional cytogenetic abnormalities in Ph-negative cells in CML patients receiving imatinib is confirmed in our series and may represent an emerging problem.32 Furthermore, this number could be underestimated because CML screening is often performed using FISH only. Although a longer follow-up observation and laboratory analyses are required to draw firm conclusions, we note that after up to 49 months follow-up the Ph-negative abnormal clone did not tend in our patients to evolve into MDS-AML, nor seems to be associated with CML clonal evolution and disease progression. Furthermore, in 4 patients the aberration was transient, suggesting the possibility of clearance of the aberrant clone over time while receiving treatment.
A registry collecting clinical and biological data regarding CML patients with Ph-negative abnormal clones has been set through the Italian GIMEMA Working Party in Chronic Myeloid Leukemia and the European Leukemia Network. We expect that prolonged observation of patients as well as collection of relevant clinical and laboratory findings in a larger series of cases may greatly improve our understanding of this still unclear phenomenon in terms of its biological or clinical significance.
We thank Prof. Francesco Lo Coco, Dr. Roberto Stasi, and Dr. James Radford Jr for critical reading and assistance preparing the article.
- 31Chromosomal abnormalities in Philadelphia chromosome-negative metaphases appearing during imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myeloid leukemia. Cancer. 2003; 98: 1905–1911., , , et al.