TO THE EDITOR
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by the presence of a specific chromosome translocation t(9;22)(q34;q11).
Molecularly, the translocation generates the BCR-ABL fusion gene encoding a BCR-ABL protein with constitutively enhanced tyrosine kinase activity. The activated BCR-ABL tyrosine kinase has been shown to be a key factor in leukemic transformation in patients as in animal model or in vitro (reviewed in Faderl et al.1).
Imatinib mesylate (Glivec™, formerly STI-571) is a rationally designed ABL′ and BCR-ABL tyrosine kinase inhibitor that has shown dramatic results in the treatment of CML. Phase II studies have demonstrated a complete hematologic response (CHR) of more than 80% and major cytogenetic response in 60% of patients that were resistant to other therapies.2 Complete cytogenetic response (CCR) has been reported in 40% of cases within 6 months but molecular response is less frequent and occurs later within 1 or 2 years after start of treatment.
Imatinib appears very effective, but its long-term effects are still unknown. Resistance may develop. Also, we and others have reported on the observation that sustained clonal chromosomal changes are found in Ph(−) mitotic cells of imatinib-treated cases (3-5). These changes are of the type often associated with MDS, that is, trisomy 8, monosomy 7, t(3;21), etc, but did not, so far, translate into active MDS or leukemia. Here again, longer follow-up is needed. This puts forward the question of the quality of remaining Ph(–) stem cells in CML patients.
We report on a Ph(–) acute leukemia (B-lineage ALL), which developed in a patient whose Ph(+) CML was in complete hematologic and cytogenetic remission after imatinib treatment.
A 44-year-old man was diagnosed with chronic phase CML after finding a leucocytosis (20 300 WBC/μl) and slight splenomegaly at a routine check up for hyperlipidemia. A Philadelphia translocation was demonstrated by cytogenetics, FISH and Southern blot.
Treatment consisted of hydroxyurea, followed by interferon-α (IFNα). After 1 year, cytogenetics showed limited response. Resistance to IFN developed progressively. IFNα was stopped and hydroxyurea used for a few weeks. Imatinib (400 mg/day) was started in July 2001, 42 months after diagnosis, and still in chronic phase. CHR was rapid. After 6 months, CCR was obtained. At the one year control, the patient was still asymptomatic but bone marrow showed 52% of lymphoblasts expressing DR (79%), CD45 (89%), CD19 (71%), CD20 (57%), CD22 (57%), CD10 (65%), CD34 (51%), CD179a intracytoplasmic (65%) and Tdt (64%). The blast cells were negative for myeloid markers, CD15, surface and intracytoplasmic immuno-globulins. The diagnosis of ALL type II of the EGIL classification was made.
Remarkably, bone marrow karyotype was normal and FISH and PCR analysis showed only residual Ph(+) cells. Imatinib was stopped and induction therapy for ALL was given consisting of cyclophosphamide, corticoid, vincristine and daunorubicin, resulting in complete remission by morphology and flow cytometry. A consolidation treatment and intrathecal methotrexate injection were given, followed by maintenance treatment, consisting of puri-nethol and dexamethasone. Currently, the patient is still in CHR with a slight thrombopenia. His marrow karyotype was still in majority Ph(−) in December 2002. An allogenic stem cell transplantation with unrelated donor is planned. At last control, before transplantation, patient was asymptomatic, but the bone marrow was again Ph(+), which suggests a return of the CML chronic phase. The patient is not receiving imatinib at the present time and thus it is not known whether these Ph(+) cells are resistant to imatinib or not.
Table 1 summarizes the results of sequential bone marrow karyotyping, FISH analysis and RQ-PCR for the presence of BCR-ABL transcripts.
Cytogenetic analyses of bone marrow cells were performed in two different laboratories at different time points, using standard methods. Whenever possible, at least 20 metaphases were analyzed at each sampling. FISH was also performed using the commercial probes LSI BCR/ABL, ES (Vysis, Downers Grove, IL, USA) and following the manufacturer's recommendations. The target DNA consisted of fixed cytogenetic cell suspension, and at least 200 nuclei were scored. The false positive rate with this probe is below 1%.
Molecular detection of BCR-ABL was also conducted in different laboratories with different methods. From November 1999 quantitative detection was conducted by real time PCR (Taqman technology). In short, total RNA was isolated from WBC lysates using Tripure reagent (Roche Molecular Biochemi-cals) and reverse transcribed using random hexamers and M-MLV Reverse Transcriptase (Roche Molecular Biochemicals), according to the manufacturer's instructions. cDNA transcribed from 100 ng of total RNA was mixed with 250 μ M of both forward (5′ IndexTermGCCACTGGATTTAAGCAGAGTTC) and reverse (5′ IndexTermTCAGACCCTGAGGCTCAAAGTC) primers and 150 μ M of the hydrolysis probe (6-FAM-5′ IndexTermAGCCCTTCAGCGGCCAGTAGCATC 3′-TAMRA). Amplification was performed in duplicate in a 25 μl volume using the Core reagent kit (Eurogentec, Belgium) in a PRISM 7700 Sequence Detector (ABI). After 2 min at 50°C, followed by 10 min at 94°C, 50 amplification cycles were performed: 95°C for 15 s and 60°C for 1 min. The β2 microglobulin gene was used as the reference gene and K562 as a positive control. The threshold cycle values (Ct) for each reaction were determined and, after normalization with the reference gene, Bcr-Abl (b2a3) chimeric transcripts were quantified against K562 by the comparative Ct method.6
After imatinib, the amount of transcripts dropped by 1 log, while at the time of ALL it increased significantly up to 22% of K562 reference, then dropped dramatically with ALL induction therapy.
In summary, we observed a CHR and CCR in a CML patient treated with imatinib in late chronic phase, resistant to IFNα. Shortly after, he presented with B-lineage ALL. The leukemic cells were clearly Ph(−), with a normal karyotype.
There are only two other cases reported of a Ph(–) acute leukemia (AML and ALL) arising after successful treatment with imatinib7 (See also, Huh HJ et al. Hematol J. 2003; 4 (Suppl. 2): 177-178 (abstract)). The fact that the leukemic blasts are Ph(−), while residual Ph(+) cells are detected by PCR, suggests that it is a secondary leukemia rather than a blast crisis of CML. The small increase in the amount of transcripts, seen in our patient concomitant with the ALL episode is related to a net increase in cellularity with less than 5% Ph(+) cells, representing the CML background.
A few other cases (three AML, two ALL) have been reported of acute leukemia in the context of CCR of CML induced by IFNα and other chemotherapy8,9,10,11,12 (Table 2). It is of concern whether this kind of evolution will be more frequent after imatinib: an unwanted side effect of successful eradication of Ph(+) clone.
The occurrence of cytogenetically abnormal clones, Ph(−) has been repeatedly reported in imatinib-treated patients.3,4,5 In our case, the karyotype was normal, which suggests that cytogenetics could underestimate the frequency of abnormal clones, Ph(−), arising in the context of imatinib-induced CCR.
In CML, depletion of Ph(+) stem cells make hematopoiesis dependent of Ph(–) stem cells, in theory nonmalignant. The finding of clonal changes of the MDS type or of acute leukemia suggests that in some patients Ph(−) stem cells could be representative of a preleukemic stage of CML with an early mutation conferring some level of genetic instability (first hit in a multiple-hit model).
Another interpretation is that the residual Ph(−) stem cells has accumulated genetic insults as a consequence of prior treatment and other environmental exposures. These lesions become apparent after depletion of Ph(+) hematopoiesis and may lead to MDS or acute leukemia at a later stage. The treatment can contribute to clonal selection. Indeed imatinib is also an inhibitor of c-Kit, the stem cell factor receptor, essential for stem cell function. Conceivably, as suggested by O'Dwyer et al,5, long-term administration of imatinib could have a suppressive effect on the normal stem cells leading to development of hypoplasia or dysplasia and providing a ‘window of opportunity’ to mutated progenitor cells, independent of c-kit for self-renewal and maturation.
In conclusion, our observation indicates strongly the need for long follow-up of patients in past and current imatinib trials, in order to see how the natural history of CML is modified, whether cure is sustained and survival prolonged as well as the impact of imatinib resistance.
Faderl S, Talpaz M, Estrov Z, O'Brien S, Kurzrock R, Kantarjian HM . The biology of chronic myeloid leukemia. N Engl J Med 1999; 341: 164–172.
Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: 645–652.
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This text presents research results of the Belgian programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The scientific responsibility is assumed by the authors.
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Wilde, SD., Rack, K., Vannuffel, P. et al. Philadelphia-negative acute lymphoblastic leukemia developing in a CML patient in imatinib mesylate-induced complete cytogenetic remission. Leukemia 17, 2046–2048 (2003). https://doi.org/10.1038/sj.leu.2403094
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