SEARCH

SEARCH BY CITATION

Keywords:

  • chronic myeloid leukemia;
  • Ph-negative clones;
  • chromosomal abnormalities;
  • imatinib

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

BACKGROUND.

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.

METHODS.

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.

RESULTS.

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.

CONCLUSIONS.

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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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.

Table 1. Clinical Data at Ph− Clones Presentation
PatientAge, yKaryotypePrior therapyDisease duration, moDuration of imatinib therapy, mo
  1. HU indicates hydroxyurea; IFN, interferon; ABMT, autologous bone marrow transplantation; PU, 6-mercaptopurine; AC, cytosine arabinoside mini-ICE, ifosfamide-aracytin-etoposide; BU, busulfan.

16546,XX[7]; 47,XX,+8[13]HU1511
   IFN  
25946,XX[11]HU3312
  47,XX,+8[5]ABMT  
  46,XX,t(9;22)(q34;q11)[1]IFN  
33446,XY[16]; 47,XY,+8[4] 1212
47446,XY[18]HU3424
  46,XY,t(6;7)(p24;q21)[2]IFN  
57746,XY,dup(1q)(q11q21)[20] 1111
64846,XX[4]HU7424
  47,XX,+8[13]IFN  
  46,XX, t(9;22)(q34;q11)[8]   
76346,XX[20]; 47,XX,+8[6]HU4824
   IFN  
   HU+PU+AC  
84546,XX[8]HU6922
  45,XX,−7[2]IFN  
  46,XX,t(9;22)(q34;q11)[10]Mini-ICE  
   IFN+AC  
94947,XX,+8 [5]HU4634
  46,XX,t(9;22)(q34;q11)[15]Mini-ICE  
   IFN  
105047,XX,+8 [22] 55
117946,XY,t(2;6)(p25;q23)[14]HU346
  46,XY,t(9;22)(q34;q11)[6]BU  
   IFN  
126846,XY[16]; 45,X0,−Y [4]HU666
   IFN  
136446,XY,t(7;9;22;)(p22;q34;q11)[6]HU10749
  46,XY,del(7)(q31)[6]IFN+AC  
  46,XY [8]   
Table 2. Changes in Ph− Clone Expression (as Percentage) During Follow-up
PatientFollow-up, mo% cells expressing Ph+ and Ph- clones at Δt from close evidence
  1. Ph indicates Philadelphia chromosome; +, positive; −, negative; ΔT, time from the first observation of the Ph-negative clone. Data reported in bold refers to patients with temporary abnormal Ph− clone, while data in italic refers to patients receiving dasatinib treatment.

128Ph+02080   
Trisomy 87027215   
ΔT6121826   
249Ph+1080100802662 
Trisomy 83080000 
ΔT61218253749 
430Ph+000    
t(6;7)20200    
ΔT61227    
538Ph+20000   
dup(1q)100100100100   
ΔT6122432   
649Ph+30504070150 
Trisomy 8571050284050 
ΔT61224303948 
749Ph+4220   
Trisomy 857636792   
ΔT6122442   
844Ph+49702525   
−71174622   
ΔT6122442   
940Ph+72832000  
Trisomy 82516385042  
ΔT69122436  
1031Ph+0200   
Trisomy 897988550   
ΔT6132431   
1140Ph+3010000  
t(2;6)709050300  
ΔT615243640  
1238Ph+20000  
−Y2815000  
ΔT69122436  
1329Ph+305592603000
del(7q)33805102320
ΔT3101215162025

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).

thumbnail image

Figure 1. Fluorescence in situ hybridization (FISH) detecting abnormal clones on metaphase and interphase chronic myeloid leukemia (CML) cells. FISH performed with locus-specific probes detecting BCR (green), ABL (red) signals on chromosomes 22 and 9; chromosome 7p12 (red) and its centromeric control (green); and chromosome 8 c-myc (red-green fusion signal). (A-1) A Ph-negative metaphase is observed (no fusion signals on chromosomes 9 and 22) showing monosomy 7 (unique red-green signal), whereas the Ph+ metaphase in (A-2) (arrows indicate the fusion signals on chromosomes 9 and 22) evidenced 2 normal chromosomes 7. (B) Interphase Ph-negative cell (no fusion signals from bcr and abl probes) showing 3 yellow signals (arrows) revealing trisomy 8.

Download figure to PowerPoint

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.

Cultured/Separated Cells

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).

Table 3. Cell Culture Growth on Mononucleated and CD34+ Bone Marrow Cells
  • CFU-GM indicates colony-forming units granulocyte macrophage; BFU-E, burst-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte-erythroid-macrophage-megakaryocitic.

  • *

    Data referred as colonies to 1 × 104 CD34+ cells.

  • Data referred as colonies to 1 × 105 mononucleated cells.

CD34-positive cells*
 Patient No.126*7813
 CFU-GM1869021*14679120
 BFU-E20014013*220198190
 CFU-GEMM750*101812
Mononucleated cells
 Patient No.1267813
 CFU-GM606910908678
 BFU-E1401438210110160
 CFU-GEMM130213

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).

Table 4. Fluorescence In Situ Hybridization Results on Unselected Bone Marrow Cells (BM) and Selected CD34+ and CD34− Cells
PatientBMPh+ clone (%)BMPh− clone (%)
CD34+CD34−CD34+CD34−
  1. + indicates positive; −, negative; Philadelphia, Philadelphia chromosome; BM, bone marrow cells.

10022152
21009298032
6501540101210
7222636560
816330526042
13020253120

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.

thumbnail image

Figure 2. Bone marrow angiogenesis. Immunohistochemical staining of vascularization of the bone marrow biopsy using CD34 monoclonal antibody. (A) Normocellular bone marrow biopsy of Patient 8 showing the presence of rare sinusoids with a thin wall consisting of an inner complete layer of flattened endothelial cells, CD34 positive. (B) Hypercellular bone marrow of a patient with myeloproliferative-myelodysplastic syndrome with increased angiogenesis.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Prof. Francesco Lo Coco, Dr. Roberto Stasi, and Dr. James Radford Jr for critical reading and assistance preparing the article.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES