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July 2002, Volume 16, Number 7, Pages 1390-1393
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Spotlight Correspondence
Clonal Ph-negative hematopoiesis in CML after therapy with imatinib mesylate is frequently characterized by trisomy 8
M K Andersen1, J Pedersen-Bjergaard1, L Kjeldsen2, I H Dufva3 and K Brøndum-Nielsen4

1Department of Clinical Genetics, The Cytogenetic Laboratory, Section of Hematology/Oncology, Rigshospitalet, Copenhagen, Denmark

2Department of Hematology, Rigshospitalet, Copenhagen, Denmark

3Department of Hematology, Amtssygehuset Herlev, Denmark

4John F Kennedy Institute, Glostrup, Denmark

Correspondence to: M K Andersen, Department of Clinical Genetics, The Cytogenetic Laboratory 4052, Section of Hematology/Oncology, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark; Fax: +45 35 45 25 77

Abstract

Leukemia (2002) 16, 1390-1393. doi:10.1038/sj.leu.2402634

TO THE EDITOR

Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph), the chromosome 22 derivative of the balanced translocation, t(9;22)(q34;q11). By fusing the ABL gene on 9q34 to the BCR gene on 22q11, the abl tyrosine kinase is constitutively activated1 causing proliferation and accumulation of immature and mature myeloid cells in blood and bone marrow. The Philadelphia chromosome and the BCR/ABL transcript are important markers in diagnosing and monitoring cytogenetic and molecular response to treatment in CML.

Therapy with conventional cytostatic agents, such as hydroxurea and busulfan, is able to normalize blood cell counts and eliminate splenomegaly. However, the Ph-positive clone persists in blood and bone marrow cells.2,3 More recent types of treatment, such as IFN and the tyrosine kinase inhibitor, imatinib mesylate, have the potential to induce cytogenetic remission with disappearance of the Ph chromosome4,5, but molecular remissions are rare.6 So far, only allogeneic bone marrow transplantation (ABMT) has been able to induce permanent molecular remission in larger series of patients with CML.7

In a few cases of Ph-positive CML, development of new cytogenetically abnormal, Ph-negative clones has been reported following therapy with IFN, in some cases administered after therapy with conventional cytostatic agents.8,9,10,11,12,13 Quite recently, Ph-negative clones have also been observed after therapy with imatinib mesylate.14,15,16 We report three such cases and review the literature of cases of CML with new Ph-negative clones emerging after therapy. The development of cytogenetically unrelated, Ph-negative clones may be much more common following therapy with imatinib mesylate than after other types of therapy, and may support the multi-step model of leukemic transformation, also in CML.

Thirty consecutive patients with Ph-positive CML were enrolled in the Novartis protocols 113 and 114 and treated with imatinib mesylate at our institutions. So far, three patients have developed cytogenetically unrelated, Ph-negative clones as reported below.

A 65-year-old woman was diagnosed with Ph-positive CML. A blood examination showed hemoglobin (Hb) 10.1 g/dl, white blood cell (WBC) 14 ´ 109/l, and platelets 2600 ´ 109/l. In order to obtain a rapid reduction of the platelet count, the patient was initially treated with hydroxurea and cytosine-arabinoside for 2 months. Therapy with IFN was initiated, but was withdrawn after only 1 month due to severe side-effects. During subsequent therapy with imatinib mesylate (Glivec, Novartis, Basel, Switzerland), 400 mg/day, in a controlled clinical trial (Novartis protocol 113, the expanded access study), the platelet count decreased to normal values and Ph-positive cells disappeared from the bone marrow as assessed by conventional cytogenetics and fluorescence in situ hybridization (FISH) (Table 1). However, persisting transfusion-dependent anemia developed (Hb 8.9 g/dl), and after 6 months of therapy with imatinib mesylate, a small Ph-negative clone with trisomy 8 was detected in bone marrow cells. After another 3 months two Ph-negative, unrelated clones were observed, one with trisomy 8, the other with monosomy 7. By then the bone marrow was hypoplastic with dysplastic features. Platelet counts were slightly reduced to 127 ´ 109/l, whereas WBC was within normal limits. The major type BCR/ABL fusion transcript detected by RT-PCR persisted in peripheral blood despite disappearance of the Ph chromosome and development of new clones. The patient continues on therapy with imatinib mesylate.

A 37-year-old man was diagnosed with Ph-positive CML. The Hb was 12.1 g/dl, WBC 71 ´ 109/l, and platelets 1493 ´ 109/l. After therapy with hydroxurea for 3 months, it was replaced by IFN. Six months after diagnosis the patient developed severe bone pain of the lower extremities, and bone marrow morphology and cytogenetic analyses indicated an accelerated phase of CML (Table 1). Therapy with hydroxurea was added to IFN, and a chronic phase of the disease was re-established. After 12 months of therapy with IFN, there was no cytogenetic response, and therapy with imatinib mesylate, 600 mg/day, was initiated in a controlled clinical trial (Novartis protocol 114). A major cytogenetic response was obtained after 10 months, but at the same time two Ph-negative clones were detected in the bone marrow, one with trisomy 8 and one with tetrasomy 8 (Table 1). The patient was moderately anemic (Hb 9.8 g/dl), whereas WBC and platelet counts were normal. Identical cytogenetic findings were observed 2 months later during continued therapy with imatinib mesylate. The patient has now been referred to ABMT.

A 62-year-old man was diagnosed with Ph-positive CML in accelerated phase. A blood count showed Hb 11.2 g/dl, WBC 134 ´ 109/l, and platelets 270 ´ 109/l. The patient was treated with hydroxurea and IFN following which blood cell counts normalized. After 10 months, therapy with IFN was withdrawn as there was no cytogenetic response (Table 1), and treatment with imatinib mesylate, 600 mg/day, was initiated in a controlled clinical trial (Novartis protocol 114). A complete cytogenetic response was obtained after 3 months of therapy, but 6 months later a new Ph-negative clone with non-recurrent structural abnormalities of chromosomes 1 and 13 appeared in the bone marrow. Blood cell counts are still within normal limits, and therapy with imatinib mesylate continues.

The development of new Ph-negative, cytogenetically unrelated clones after therapy of Ph-positive CML with imatinib mesylate is a new phenomenon,14,15,16 but a few similar cases have previously been observed after therapy with IFN.8,9,10,11,12,13 In total, at least 17 cases of Ph-positive CML developing new Ph-negative clones after therapy with IFN or imatinib mesylate have been reported in the literature (Table 2). Eleven cases presented a new Ph-negative clone with trisomy 8, and two of these cases also had another Ph-negative unrelated clone with monosomy 7 or del(7q). Interestingly, in one case,8 trisomy 8 was present in a Ph-negative as well as in a Ph-positive clone, and another patient developed tetrasomy 8 (case 2 in the present study).

One important question is whether the Ph-negative clones represent development of a new leukemic complication, either a de novo disease or a therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/t-AML), or whether they represent a new manifestation of the original disease. Another question is whether the cytogenetically abnormal, Ph-negative clones predict or directly indicate development of myelodysplasia or acute myeloid leukemia, or whether they represent a more benign, perhaps transient phenomenon.

It seems very unlikely that so many patients with Ph-positive CML within a rather short period of observation, should develop new de novo leukemias. In particular, it seems unlikely that patients, such as our case 1 and No. 3 reported by Gambacorti-Passeri et al.,14 should develop two different, new leukemia-like diseases, one with trisomy 8, the other with monosomy 7 or del(7q).

It also seems unlikely that the new clones represent cases of t-MDS/t-AML. Some of the patients with Ph-negative, cytogenetically unrelated clones had not previously received chemotherapy known to be leukemogenic. Alkylating agents had only been administered briefly in five patients, and topoisomerase II inhibitors had not been administered in any of the reported cases (Table 2). Only two cases, including case 1 in the present study, presented monosomy 7 or deletion of 7q highly characteristic of therapy-related leukemia, whereas all other new Ph-negative clones showed chromosome aberrations uncharacteristic of t-MDS or t-AML. Finally, there is no biological reason to suspect imatinib mesylate to be leukemogenic.

Most likely, the development of new Ph-negative clones reflects the existence of a Ph-negative preleukemic stage of CML. Cells at this stage subsequently acquire the Ph chromosome, but after therapy mainly targeting Ph-positive cells, the Ph-negative cells reappear with other genetic changes visible as new cytogenetically unrelated clones. Previously, Ph-negative, but clonal leukemic progenitor cells have been demonstrated in Ph-positive CML. Thus, Ph-negative cells cultured from the blood of patients with Ph-positive CML have showed a skewed expression of glucose-6-phosphate dehydrogenase (G6PD) in female heterozygotes indicating a clonal origin.17,18 Likewise, clonal Ph-negative B lymphocytes have been demonstrated based on the expression of a single G6PD phenotype in Ph-positive CML.19 In addition, cases of Ph-negative CML acquiring the Ph chromosome later during the course of the disease have been reported.20

As trisomy 8 is a highly characteristic, evolutionary aberration in Ph-positive CML, the observation of trisomy 8 as a new clonal abnormality in 11 out of 17 patients in Ph-negative clones supports the new clone as being derived from the original disease. Furthermore, the observation of three different clones, one with t(9;22) only, another with trisomy 8 only, and a third clone with t(9;22) plus trisomy 8 in a patient treated with IFN,8 also suggests a common origin of the Ph-negative and the Ph-positive clones in CML.

Previously, trisomy 8 was regarded as a rather unimportant chromosome aberration in AML, often observed only in subclones or as an evolutionary event. Recently, however, a specific gene expression profile has been observed in cases of AML with trisomy 8 as a sole aberration compared to cases with a normal karyotype. Genes associated with apoptosis were down-regulated, and genes located to chromosome 8 up-regulated, possibly due to a gene dose-effect. The observation of a clone with tetrasomy 8 in case No. 2 in the present study supports a similar gene dose-effect of supernumerary chromosomes 8, also in CML.

The fact that eight cases of CML presented new Ph-negative, cytogenetically unrelated clones shortly after the clinical introduction of imatinib mesylate, suggests that the development of new clones could be particularly common following therapy with this drug. This is confirmed by our observation that three out of 30 consecutive patients, so far, have developed cytogenetically unrelated Ph-negative clones after therapy with imatinib mesylate. Based on the specific mechanism of action of imatinib mesylate, targeting only Ph-positive cells, this observation may not be unexpected. As imatinib mesylate specifically eradicates Ph-positive cells, the Ph-negative leukemic progenitors may survive, expand selectively, and by clonal evolution present as cytogenetically unrelated clones.

In the few cases of CML developing Ph-negative, cytogenetically unrelated clones observed so far (Table 1), the follow-up period has been short (1-23 months). Two patients developed AML,11,13 and two patients developed refractory cytopenia or dysplastic changes in the bone marrow consistent with myelodysplasia at the time of the development of the new clone (Ref. 10 and case No. 1 in the present study). In the remaining 13 cases, the new Ph-negative clones evolved during the chronic phase of CML, and in three cases the new Ph-negative clones were reported as transient.9,13 In conclusion, the clinical significance of the new phenomenon remains to be further clarified. In analogy with chronic phase CML, in which cytogenetic abnormalities in addition to t(9;22) predicts deterioration of the disease, the Ph-negative unrelated clones must be suspected in the long term to reflect loss of control of the disease.

References

1 Kalidas M, Kantarjian H, Talpaz M. Chronic myeloid leukemia. JAMA 2001; 286: 895-898. MEDLINE

2 Hehlmann R, Heimpel H, Hasford J, Kolb HJ, Pralle H, Hossfeld DK, Queisser W, Loffler H, Heinze B, Georgii A. Randomized comparison of busulfan and hydroxurea in chronic myelogeneous leukaemia: prolongation of survival by hydroxurea. The German CML Study Group. Blood 1993; 82: 398-407. MEDLINE

3 Hehlman R, Heimpel H, Hasford J, Kolb HJ, Pralle H, Hossfeld DK, Queisser W, Loffler H, Hochhaus A, Heinze B. Randomized comparison of interferon-alpha with busulfan and hydroxurea in chronic myelogeneous leukaemia. The German CML Study Group. Blood 1994; 84: 4064-4077. MEDLINE

4 Talpaz M, Kantarjian HM, McCredie K, Trujillo JM, Keating MJ, Gutterman JU. Hematologic remission and cytogenetic improvement induced by recombinant human interferon alpha A in chronic myelogeneous leukemia. N Engl J Med 1986; 314: 1065-1069. MEDLINE

5 Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031-1037. MEDLINE

6 Mahon FX, Delbrel X, Cony-Makhoul P, Faberes C, Boiron JM, Barthe C, Bilhou-Nabera C, Pigneux A, Mait G, Reiffers J. Follow-up of complete cytogenetic remission in patients with chronic myeloid leukemia after cessation of interferon alfa. J Clin Oncol 2002; 20: 214-220. MEDLINE

7 Horowitz MM, Rowlings PA, Passweg JR. Allogeneic bone marrow transplantation for CML: a report from the International Bone Marrow Transplant Registry. Bone Marrow Transplant 1996; 17 (Suppl. 3): S5-S6. MEDLINE

8 Casali M, Truglio F, Milone G, Di Raimondo F, Parrinello G, Maserati E, Pasquali F. Trisomy 8 in Philadelphia chromosome (Ph1)-negative cells in the course of Ph1-positive chronic myelocytic leukemia. Genes Chromosomes Cancer 1992; 4: 269-270. MEDLINE

9 Bilhou-Nabera C, Marit G, Devianne I, Viard F, Salzes S, Montastruc M, Renoux M, Broustet A, Reiffers J, Bernard P. A second case of trisomy 8 in Philadelphia chromosome (Ph)-negative cells during the course of Ph-positive chronic myelocytic leukemia. Genes Chromosomes Cancer 1993; 4: 255-256.

10 Ariyama T, Inazawa J, Uemura Y, Kakazu N, Maekawa T, Urase F, Irimajiri K, Horiuchi A, Nakamura Y, Abe T. Clonal origin of Philadelphia chromosome negative cells with trisomy 8 appearing during the course of alpha-interferon therapy for Ph positive chronic myelocytic leukemia. Cancer Genet Cytogenet 1995; 81: 20-23. MEDLINE

11 Ohtsuka E, Kikuchi H, Abe Y, Moriyama K, Ohno E, Hirota K, Tezono K, Nasu M. Acute myeloblastic leukaemia without Philadelphia chromosome developing after interferon therapy for chronic myelocytic leukaemia with Philadelphia chromosome. Br J Haematol 1995; 90: 951-953. MEDLINE

12 Izumi T, Imagawa S, Hatake K, Miura Y, Ariyama T, Inazawa J, Abe T. Philadelphia chromosome-negative cells with trisomy 8 after busulfan and interferon treatment of Ph1-positive chronic myelogenous leukemia. Int J Hematol 1996; 64: 73-77. MEDLINE

13 Fayad L, Kantarjian H, O'Brien S, Seong D, Keating M, Talpaz M. Emergence of new clonal abnormalities following interferon-alpha induced complete cytogenetic response in patients with chronic myeloid leukemia: report of three cases. Leukemia 1997; 11: 767-771. MEDLINE

14 Marktel S, Bua M, Marin D, Chase A, Udom C, Armstrong L, Apperley JF, Olavarria E, Goldman JM. Emergence of additional chromosomal abnormalities following treatment with STI571 (Imatinib Mesylate) for Philadelphia positive chronic myeloid leukemia in chronic phase. Blood 2001; 98: 617a, (Abstra. 2584).

15 Gambacorti-Passeri C, Giudici G, le Coutre P, Bungaro S, Pogliani EM, Kreuzer K-A, Tornaghi L, Alberti D, Corneo G, Biondi A. Non random chromosomal abnormalities in Ph-negative bone marrow (BM) cells from CML patients achieving major cytogenetic responses (MCR) with STI571 (GleevecÔ). Blood 2001; 98: 257b, (Abstr. 4762).

16 Meunier V, Giraudier S, Van den Akker J, Perot C, Jouault H, Caron-Servan B, Robin V, Tulliez M, Imbert M. Treatment with STI571 (NovartisÒ) in chronic myeloid leukemia (CML): unexpected hematological findings in a series of 26 patients. Blood 2001; 98: 263b, (Abstr. 4784).

17 Fialkow PJ, Martin PJ, Najfeld V, Penfold GK, Jacobsen RJ, Hansen JA. Evidence for a multistep pathogenesis of chronic myelogeneous leukemia. Blood 1981; 58: 158-163. MEDLINE

18 Raskind WH, Ferraris AM, Najfeld V, Jacobsen RJ, Moohr JW, Fialkow PJ. Further evidence of a clonal Ph-negative stage in some cases of Ph-positive chronic myelocytic leukemia. Leukemia 1993; 7: 1163-1167. MEDLINE

19 Ferraris AM, Canepa L, Melani C, Miglino M, Broccia G, Gaetani GF. Clonal B lymphocytes lack bcr rearrangement in Ph-positive chronic myelogenous leukaemia. Br J Haematol 1989; 73: 48-50. MEDLINE

20 Lisker R, Casas L, Mutchinick O, Perez-Chavez F, Labardini J. Late-appearing Philadelphia chromosome in two patients with chronic myelogenous leukemia. Blood 1980; 56: 812-814. MEDLINE

21 Virtaneva K, Wright FA, Tanner SM, Yuan B, Lemon WJ, Caligiuri MA, Bloomfield CD, de La Chapelle A, Krahe R. Expression profiling reveals biological differences in acute myeloid leukemia with isolated trisomy 8 and normal cytogenetics. Proc Natl Acad Sci USA 2001; 98: 1124-1129. MEDLINE

Tables

Table 1 Cytogenetic evolution in three CML patients treated with imatinib mesylate

Table 2 A review of the literature of cases with Ph-positive CML developing new Ph-negative clones

Received 23 April 2002; accepted 23 April 2002
July 2002, Volume 16, Number 7, Pages 1390-1393
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