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Chromosomal abnormalities in Philadelphia chromosome-negative metaphases appearing during imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase
Version of Record online: 17 SEP 2003
Copyright © 2003 American Cancer Society
Volume 98, Issue 9, pages 1905–1911, 1 November 2003
How to Cite
Medina, J., Kantarjian, H., Talpaz, M., O'Brien, S., Garcia-Manero, G., Giles, F., Rios, M. B., Hayes, K. and Cortes, J. (2003), Chromosomal abnormalities in Philadelphia chromosome-negative metaphases appearing during imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase. Cancer, 98: 1905–1911. doi: 10.1002/cncr.11729
- Issue online: 22 OCT 2003
- Version of Record online: 17 SEP 2003
- Manuscript Accepted: 25 JUL 2003
- Manuscript Revised: 15 MAY 2003
- Manuscript Received: 20 FEB 2003
- chronic myelogenous leukemia (CML);
- Philadelphia chromosome;
- clonal evolution
Anecdotal cases of chromosomal abnormalities in Philadelphia chromosome (Ph)-negative metaphases have been reported in patients with chronic myelogenous leukemia (CML) in the chronic phase during treatment with interferon and, more recently, with imatinib. This phenomenon is different from true clonal evolution in that the additional cytogenetic abnormality occurs in Ph-negative cells.
The authors analyzed their experience with 342 patients with CML in chronic phase treated with imatinib to investigate the frequency and significance of this event.
After a median follow-up of 30 months (range, 16–35 months), 21 patients (6%; 95% confidence interval, 0.04, 0.09) developed 25 chromosomal abnormalities in Ph-negative cells. Thirteen (54%) of these abnormalities were seen in 2 or more metaphases. The median time from the start of treatment with imatinib to the appearance of the abnormalities was 6 months (range, 3–22 months). The most common cytogenetic abnormality detected was trisomy 8 (33%). Twenty of 21 patients (95%) achieved a major (Ph < 35%) cytogenetic response (complete cytogenetic response in 13–62%). After a median follow-up of 22 months (range, 4–33 months), all 21 patients were alive, 20 of them in chronic phase and in complete hematologic response. None of the patients showed features of myelodysplasia.
Cytogenetic abnormalities occur in Ph-negative cells in a fraction of patients with CML in chronic phase treated with imatinib. With a short follow-up, no clear clinical consequences can be identified. Cancer 2003. © 2003 American Cancer Society.
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by a specific cytogenetic abnormality, the Philadelphia chromosome (Ph). This is a balanced translocation, involving a fusion of the Abelson oncogene (ABL) from chromosome 9q34 with the breakpoint cluster region (BCR) on chromosome 22q11.2, t(9;22)(q34;q11.2). The molecular consequence of this translocation is the generation of a BCR-ABL fusion oncogene, which translates into a Bcr-Abl oncoprotein of 210 kilodalton (kD) molecular weight, p210Bcr/Abl, with increased tyrosine kinase activity.1 This tyrosine kynase activity of the Bcr-Abl protein is essential to its transforming capability as well as its increased binding to the actin cytoskeleton.2
CML has a biphase or triphase clinical course. Approximately 90% of patients are diagnosed in the chronic phase, but the disease eventually evolves to a blastic phase unless successfully treated. Approximately two-thirds of patients manifest an accelerated phase. A distinct feature of disease progression is the appearance of additional cytogenetic abnormalities in the Ph-positive cells.3 This phenomenon, known as clonal evolution, frequently involves a second Ph, trisomy of chromosome 8, and isochromosome 17 and other abnormalities of chromosome 17,3 although other abnormalities have been described. Clonal evolution is considered a criterion of accelerated phase, although when it represents the only criterion of transformation, it is associated with a better prognosis than other criteria of accelerated phase.4–6
Standard therapy for CML has included allogeneic stem cell transplantation and interferon-alpha (IFN-α). More recently, imanitib mesylate (STI571, Gleevec, Novartis, East Hanover, NJ) has demonstrated significant activity in CML.7 Imatinib is a potent tyrosine kinase inhibitor selective for Abl, Bcr/Abl, c-kit, and platelet-derived growth factor receptor.8 Approximately 65% of patients with CML in the chronic phase who failed IFN-α therapy achieved a major cytogenetic response.7, 9 Among patients with clonal evolution and no other criteria for accelerated phase, 50–70% may achieve a major cytogenetic response.5, 6
During the course of therapy with IFN-α, cytogenetic abnormalities have been reported to occur in the Ph-negative cells.10 Isolated examples of a similar phenomenon have been reported in patients treated with imatinib.11–14 We analyzed our experience in patients with CML treated with imatinib in chronic phase to determine the frequency and clinical significance of the appearance of cytogenetic abnormalities in Ph-negative cells.
MATERIALS AND METHODS
Patients with Ph-positive CML treated with imatinib at M. D. Anderson Cancer Center (Houston, TX) were analyzed. Patients were eligible for the current analysis regardless of whether they received imatinib after IFN-α failure or as their first line of therapy. Patients with clonal evolution at the initiation of therapy with imatinib were excluded from the current study. Patients analyzed were included in 1 of 3 studies: ID01-151, a study of high-dose imatinib (800 mg/day) for patients with previously untreated, early chronic phase CML; ID01-015, a study of standard-dose imatinib (400 mg/day) for patients with previously untreated CML in the chronic phase; and DM99-367, a study of imatinib (400 mg/day) for patients with CML in chronic phase after IFN-α failure. Patients in the DM99-367 study were part of a multicenter, multinational study (Novartis study 110). Eligibility criteria and pretreatment and follow-up studies were as previously described.7, 9, 15
For the current study, we reviewed all the cytogenetic analyses of patients during the course of therapy. Cytogenetic analysis, using the G-banding technique, was performed in all patients before the initiation of therapy and every 3 months during the course of therapy. Cytogenetic response was determined based on the percentage of Ph-positive metaphases as previously described.7, 9, 15
Between December 1999 and March 2002, 342 patients with chronic-phase CML were treated with imatinib. Among them, 237 patients (69%) had CML after failure to IFN-α therapy and 105 (31%) were previously untreated. Among previously untreated patients, 50 (48%) were treated with a daily dose of 400 mg of imatinib and 55 (52%) were treated with 800 mg. After a median follow-up of 30 months, 21 patients (6%; 95% confidence interval [95% CI], 0.04–0.09) had developed 25 chromosomal abnormalities in Ph-negative metaphases during treatment. By definition, this phenomenon was evaluable only among patients who achieved any cytogenetic response (i.e., < 95% Ph metaphases). Thus, 21 of 272 of evaluable patients (8%; 95% CI, 0.05–0.12) developed at least 1 of these abnormalities. Four patients had two different abnormalities presenting at different times. The clinical characteristics of all 21 patients are shown in Table 1. The median age of the patients was 52 years (range, 25–75 years) and 8 patients (38%) were females. Nineteen patients (90%) had failed previous IFN-α therapy (hematologic failure in 2 patients, cytogenetic failure in 11 patients, and intolerance in 6 patients) and 2 (9%) were previously untreated (Patients 8 and 9, Fig. 1B). Eighteen (95%) of the 19 previously treated patients who had received IFN-α achieved a major cytogenetic response (complete cytogenetic response in 13) as their best cytogenetic response to imatinib and the 2 untreated patients achieved a partial cytogenetic response. None of the 50 patients treated with a dose of 800 mg daily who were analyzed for the current study had developed these abnormalities at the time of last follow-up. The additional cytogenetic abnormalities appeared after a median of 6 months (range, 3–22 months) from the initiation of imatinib therapy. At the time these abnormalities were first noted, 15 patients (71%) had achieved a major cytogenetic response (6 complete and 9 partial responses) and 6 patients (29%) had achieved a minor response. The most common cytogenetic abnormalities were trisomy 8 in 8 patients (38%), monosomy 7 in 4 patients (19%), deletion 20q- in 3 patients (14%), and monosomy 5 in 2 patients (9%; Table 2). The median number of metaphases involved (of 20 analyzed) was 2 (range, 1–20 metaphases). Twelve of the abnormalities (48%) in 8 patients involved only 1 metaphase and were considered nonclonal. However, in at least 6 of the 13 patients with clonal abnormalities (i.e., 2 or more metaphases were involved), a cytogenetic analysis showing only one abnormal metaphase was followed or preceded in other analyses by 2 or more metaphases with the same abnormality.
|Characteristics||Median (range)||No. (%)|
|Male gender||13 (62)|
|Age (yrs)||52 (25–75)|
|Hemoglobin level (g/dL)||11.6 (9.6–14.3)|
|Platelet count (× 109/L)||197 (103–1074)|
|Leukocyte count (× 109/L)||6.5 (3.5–45.8)|
|Basophils in PB (%)||1 (0–6)|
|Blasts in PB (%)||0 (0–2)|
|Blasts in BM (%)||1 (0–6)|
|Basophils in BM (%)||1 (0–3)|
|Creatinine level (mg/dL)||0.9 (0.6–2.2)|
|Bilirubin level (mg/dL)||0.6 (0.3–1.2)|
|ALT level (IU/L)||24 (12–95)|
|Albumin level (g/dL)||4 (3.5–4.6)|
|Ph-positive metaphases (%)||100 (15–100)|
|Karyotype||Overall (%)||Clonal (%)a||Nonclonal (%)|
|+8||8 (32)||6 (46)||2 (17)|
|−7||4 (16)||2 (15)||2 (17)|
|20q−||3 (12)||2 (15)||1 (8)|
|−5||2 (8)||—||2 (17)|
|7q−||2 (8)||1 (8)||1 (8)|
|4p+||1 (4)||—||1 (8)|
|5q−/21p+||1 (4)||1 (8)||—|
|add 12/del 18||1 (4)||1 (8)||—|
|−4, −6, −8, −21||1 (4)||—||1 (8)|
|t(1; 7)||1 (4)||—||1 (8)|
|t(9; 14)||1 (4)||—||1 (8)|
These cytogenetic abnormalities were frequently transient. Figure 1 shows the sequence of the cytogenetic studies in the patients with clonal abnormalities. Twelve of these abnormalities (48%) have been observed only once (11 involving 1 metaphase). The remaining 12 abnormalities (50%) have been seen in a median of 2 analyses (range, 2–8), although not always consecutively. In three patients (Patients 1, 5, and 9), there was at least one analysis that did not demonstrate the additional cytogenetic abnormality between two analyses with the additional abnormalities (although in one of them, Patient 9, trisomy 8 was identified by fluorescent in situ hybridization when the cytogenetic analysis failed to identify the abnormality). At the time of last follow-up, three patients had developed clonal evolution (i.e., additional chromosomal abnormalities within the Ph-positive clone) in addition to the cytogenetic abnormality in the Ph-negative clone. Clonal evolution was comprised of trisomy 8 in all cases. The cytogenetic abnormality identified in the Ph-negative cells in these patients was trisomy 8 in two patients and deletion of 7q in one.
Table 3 shows the cytogenetic response at different times. Four (50%) of 8 patients with a partial cytogenetic response at the time the additional abnormality was discovered improved their cytogenetic response to a complete cytogenetic response. These four patients had trisomy 8 (Patient 1, Fig. 1C), del 20q- (Patient 5, Fig. 1A), monosomy 7 (Patient 8, Fig. 1C), and pseudiploid 5q-/21p+ (Patient 6, Fig. 1A) in the Ph-negative cells, respectively. In two of these patients (Patients 5, 6; Fig. 1A), the additional chromosomal abnormalities were still noted in at least one analysis after achieving a complete cytogenetic response. Patient 1 (Fig. 1C) also was found to have a transient clonal evolution with trisomy 8 in Ph-positive cells. Both abnormalities were transient and at the time of last follow-up the patient had lost the complete cytogenetic response. In contrast, 4 (19%) patients had lost their cytogenetic response at the time of last follow-up. Three patients with a partial cytogenetic response at the time abnormalities were identified in the diploid metaphases progressed to a minor cytogenetic response (12.5–58% Ph, 15–55% Ph positive, and 15–40% Ph positive, respectively). Two patients who had acheieved a complete cytogenetic response at the time these abnormalities were noted had a partial response (5% and 9.5% Ph-positive metaphases, respectively) at the time of last follow-up. After a median follow-up time of 30 months (range, 16–35 months) from the initiation of imatinib therapy and 22 months (range, 4–33 months) from the detection of the chromosomal abnormality, all patients were alive, 20 of whom (95%) were in the chronic phase and in complete hematologic response and 1 in accelerated phase (clonal evolution).
|Characteristics||Complete (%)||Partial (%)||Minor (%)|
|Best response||13 (62)||8 (38)||—|
|Time of additional abnormality||8 (38)||8 (38)||5 (24)|
|Last follow-up||10 (48)||7 (33)||4 (19)|
No evidence of dysplasia had been identified in any of these patients at the time of, or after, the detection of these abnormalities. Three patients developed Grade 3 (NCI CTC version 2.0) or worse myelosuppression during therapy: thrombocytopenia in two and neutropenia and thrombocytopenia in one. The myelosuppression was observed before the abnormalities were detected in all three patients. A dose reduction (from 400 mg to 300 mg daily) was required in 3 patients. Two of these patients were eventually able to tolerate an increase in their dose from 300 mg to 400 mg (for 1 patient, the dose later was increased to 600 mg) after 9 and 12 months, respectively. The third patient was given granulocyte–colony-stimulating factor to enable continuation of treatment with the original dose (400 mg/day) and, eventually, an increase to 600 mg/day.
The development of chromosomal abnormalities in the Ph-negative metaphases during treatment in patients with CML has been recognized in the past. Before effective therapy for CML was available, treatment with busulphan or hydroxyurea resulted in hematologic but not in cytogenetic responses. Thus, this phenomenon could not be evaluated. With IFN-α-based therapy, 30–70% of patients achieved a cytogenetic response, which was major in 20–50%.1 Fayad et al.10 first reported the occurrence of cytogenetic abnormalities in the Ph-negative cells in three patients responding to IFN-α. One of these patients developed 5q- in one analysis and a complex karyotype in a subsequent analysis, the second developed +18p11, and the third developed deletion 11q21. The first patient developed a myelodysplastic syndrome 86 months later and the other 2 patients remained in complete hematologic and cytogenetic response at the time of the report (6 months and 33 months, respectively) after these abnormalities first were noted.10
There are anecdotal cases and small series in the literature reporting the development of chromosomal abnormalities in Ph-negative cells after imatinib therapy.11–14 The pathogenesis and clinical significance of these abnormalities is still unclear. In the current study, we report the largest series of patients who have developed this phenomenon to better understand the scope and significance of these changes. After a median follow-up of 30 months, only 8% of patients who achieved a cytogenetic response (i.e., had evaluable Ph-negative metaphases) developed any abnormality. This was clonal in 62% of patients and persisted (i.e., in more than 1 analysis) in 52% of patients. The majority of the abnormalities reported in the current study and elsewhere were chromosome changes frequently associated with myelodysplastic syndromes. However, at the time of last follow-up, none of the current study patients had developed evidence of myelodysplasia with repeated bone marrow analysis. This is in contrast to the study by Bumm et al.,11 in which 8 patients, representing 17% of all patients analyzed, developed similar abnormalities (including 2 patients whose only abnormality was loss of the Y chromosome). Two of the patients in their report had developed myelodysplasia.11 Because these patients will likely require long-term therapy with imatinib, close follow-up of patients is important.
A unique feature of our study is that cytogenetic abnormalities were identified in the Ph-negative metaphases in two previously untreated patients. To date, all patients reported have been previously treated.11–14 Previous treatment with idarubicin and cytosine arabinoside (Ara-C) has been suggested as a risk factor.11 Most patients at our institution have been treated with Ara-C in combination with IFN-α for more than 10 years. The majority of patients who have failed to respond to IFN-α have received other agents, most frequently homoharringtonine before imatinib became available. Therefore, the role of previous exposure to these agents is difficult to dissect from the exposure to IFN-α itself. However, the finding in our two previously untreated patients and the historic observation in patients treated with IFN-α alone10 suggest that the clonal abnormality may be intrinsic to the primitive hematopoietic stem cell and may not be therapy related. Another important observation is that none of the 55 patients treated with high-dose imatinib (i.e., 800 mg/day) has developed these abnormalities. Although few patients have so far received the higher doses, it is possible that a faster and more effective elimination of the Ph malignant clone, as noted with high-dose imatinib,16 may decrease the risk of development of chromosomal abnormalities in the Ph-negative stem cells. Longer follow-up on a larger population is required to clarify this issue further.
Trisomy 8 was particularly common, representing 38% of all abnormalities identified in the current study. This abnormality has also been associated with clonal evolution.3, 4, 17 It is noteworthy that three patients in our series also developed this abnormality as clonal evolution (i.e., within the Ph-positive clone). Two of them also had this abnormality present in Ph-negative cells. C-myc is coded in chromosome 8. Jennings and Mills18 have suggested a correlation between levels of c-myc and trisomy 8 among patients who developed clonal evolution. The significance of this association when trisomy 8 presents in the Ph-negative cells is unclear.
In the current analysis, we included eight patients with abnormalities in only one metaphase. Although these were considered nonclonal, we included them for several reasons. First, because this is a newly recognized phenomenon and because an increasing number of patients are being treated for longer periods of time, the significance of even these nonclonal abnormalities requires recognition and close monitoring. Second, as an example of this uncertainty, 6 of the patients had abnormalities detected in only 1 of 20 metaphases at some point, but repeated analyses showed 2 or more metaphases with the same abnormality. This suggests that at least some of these abnormalities might represent true clones detected earlier because of the frequent monitoring. Furthermore, at least 2 patients (Patient 2, Fig. 1A; Patient 12, Fig. 1B) had a clonal abnormality (monosomy 7 in 15 metaphases and trisomy 8 in 7 metaphases, respectively) that disappeared but was followed by the appearance of a different abnormality (+8, and 7q-, respectively) in only 1 metaphase in the last follow-up. Also, one patient had two different abnormalities detected in only one metaphase in two consecutive analyses (Patient 8, Fig. 1C). Finally, most of the abnormalities reported, including the nonclonal abnormalities, frequently are seen in myelodysplastic syndromes,19 and the nonclonal abnormalities were very constant. Thus, the long-term implications might be significant and should be followed closely.
With a relatively short follow-up, it is clear that these abnormalities can be transient in some patients, even when patients continue in cytogenetic remission. Others show persistence and occasionally increase percentage of these abnormalities. Within the limitations of a small series and a relatively short follow-up period, no prognostic impact can be detected in the duration of cytogenetic remission, transformation to accelerated or blastic phase, or overall survival. Development of true clonal evolution occurred in 3 of 21 patients (14%).
The significance of these abnormalities is unclear at present. It has been suggested that treatment with imatinib may favor the manifestation of Ph-negative clonal disorders in some patients.11 Alternatively, this could support a multistep pathogenesis in CML as has been proposed for more than 20 years.20 Fialkow et al.20 established Ph-negative B-lymphoid cell lines from a patient with Ph-positive CML heterozygous for glucose-6-phosphate-dehydrogenase. The observed ratio of Type B to Type A glucose-6-phosphate-dehydrogenase was 18:45, compared with a 1:1 ratio in normal tissues (P < 0.0001). It is interesting to note that 8 of 33 evaluable Ph-negative B-lymphoid cell lines with the Type B enzyme had chromosomal abnormalities, compared with 0 of 14 Type A lines.20 This suggests that some cells from the malignant clone do not have the Ph, and yet have other abnormalities, suggesting that Ph is not sufficient for malignant transformation. Because imatinib would only affect cells expressing Ph and its tyrosine kinase, the appearance of abnormalities in Ph-negative cells may uncover some of these cells that only express the “first hit.” Another possibility is an inherent predisposition of the normal hematopoietic stem cell to develop other cytogenetic abnormalities in patients who develop Ph-positive cells. In untreated patients, these abnormalities are first overwhelmed by the presence of the Ph, which have a growth advantage but later appear upon transformation to the accelerated or blast phase within the Ph-positive clone. The efficacy of IFN-α first in suppressing the Ph-positive clone may have unmasked this phenomenon (i.e., the susceptibility of the normal stem cell to develop additional chromosomal changes). With significantly more patients achieving complete cytogenetic responses with imatinib, this phenomenon has become more evident. Whether these changes have the same implications as in myelodysplastic syndromes, i.e., to establish clonal clinically relevant disorders, or whether they are transient remains to be defined. If these changes are related to the primitive stem cell inherent fragility, it might be expected that patients who live longer because of successful treatment with imatinib may develop other diseases such as myelodysplastic syndrome. A similar phenomenon has been observed in the past, once hydroxyurea and busulfan effectively controlled the hematologic manifestations of the chronic phase of CML. Investigators then found that patients died, not from complications of chronic phase, but from a picture of acute leukemia. Initially, this was believed to be secondary to treatment. We now recognize that this acute leukemia, or blastic phase, is part of the natural history of the disease. Thus, imatinib may be now unmasking the inherent chromosomal instability in the normal stem cell that leads to the development of the Ph as well as other cytogenetic abnormalities in Ph-negative cells, the clinical relevance of which remains to be determined.
Cytogenetic abnormalities in the Ph-negative metaphases might appear in some patients with chronic-phase CML who respond to imatinib. This observation highlights the need for close monitoring of patients with CML who receive imatinib. The long-term significance of these abnormalities remains to be determined.
- 13The emergence of Philadelphia chromosome negative, trisomy 8 positive cells in patients with chronic myelogenous leukemia treated with imatinib mesylate: clinical evidence for a multistep pathogenesis [abstract]. Blood. 2002; 100: 585a., , , et al.
- 14Emergence of additional chromosomal abnormalities following treatment with STI571 (imatinib mesylate) for Philadelphia positive chronic myeloid leukemia in chronic phase [abstract]. Blood. 2001; 98: 617a., , , et al.
- 16High rates of major cytogenetic response in patients with newly diagnosed chronic myeloid leukemia in early chronic phase treated with imatinib at 400 mg or 800 mg daily [abstract]. Blood. 2002; 100: 95a., , , et al.