Clonal Aberrations in Philadelphia Chromosome Negative Hematopoiesis in Patients with Chronic Myeloid Leukemia Treated with Imatinib Or Interferon Alpha

Spotlight Correspondence 460 number. This view is supported by the results from hierarchical Supplementary Information cluster analyses including different numbers of genes with heterogenous expression, which were selected according to Supplementary Information accompanies the paper on the defined criteria (Figure 1). We found several genes that were Leukemia website (http://www.nature.com/leu). heterogenously expressed in our samples indicating that a heterogenous alteration of gene expression by imatinib could be possible. However, we could not identify groups that were References associated with imatinib treatment supporting the results from the SAM algorithm that there were no uniform imatinib-induced 1 Bumm T, Muller C, Al-Ali HK, Krohn K, Shepherd P, Schmidt E changes in gene expression. et al. Emergence of clonal cytogenetic abnormalities in PhÀ cells in some CML patients in cytogenetic remission to imatinib but In conclusion, after reaching major molecular remission restoration during first-line treatment with 400 mg imatinib per day, no of polyclonal hematopoiesis in the majority. Blood 2003; 101: uniform influence of the tyrosine kinase inhibitor on gene 1941–1949. expression patterns in Ph-negative CD34 þ stem and progenitor 2 Yuan ZM, Shioya H, Ishiko T, Sun X, Gu J, Huang YY et al. p73 is cells was observed in vivo. Therefore, based on our gene regulated by tyrosine kinase c-Abl in the apoptotic response to expression data we found no evidence for a major functional DNA damage. Nature 1999; 399: 814–817. 3 Lyman SD, Jacobsen SE. C-kit ligand and Flt3 ligand: stem/ disturbance of Ph-negative hematopoiesis using imatinib as first- progenitor cell factors with overlapping yet distinct activities. line therapy in CML. Blood 1998; 91: 1101–1134. 4 Andersen MK, Pedersen-Bjergaard J, Kjeldsen L, Dufva IH, SPOTLIGHT Brondum-Nielsen K. Clonal Ph-negative hematopoiesis in CML Acknowledgements after therapy with imatinib mesylate is frequently characterized by trisomy 8. Leukemia 2002; 16: 1390–1393. This work was supported by the Leuka¨mie Liga e.V., Du¨sseldorf. 5 Terre C, Eclache V, Rousselot P, Imbert M, Charrin C, Gervais C et al. France Intergroupe pour la Leucemie Myeloide Chronique. F Neumann1 1Department of Hematology, Report of 34 patients with clonal chromosomal abnormalities N Teutsch1 Oncology and Clinical Immunology, in Philadelphia-negative cells during imatinib treatment of S Kliszewski1 Heinrich Heine University, Philadelphia-positive chronic myeloid leukemia. Leukemia 2004; S Bork1 Du¨sseldorf, Germany; 18: 1340–1346. 2 U Steidl1 Theoretical Bioinformatics, 6 Steidl U, Kronenwett R, Rohr U-P, Fenk R, Kliszewski S, Maercker B Brors2 German Cancer Research Centre, C et al. Gene expression profiling identifies significant differences 1 Heidelberg, Germany; and between the molecular phenotypes of bone marrow-derived and N Schimkus 3 circulating human CD34+ hematopoietic stem cells. Blood 2002; N Roes1 Department of Human 1 Genetics and Anthropology, 99: 2037–2044. U Germing 7 Huber W, von Heydebreck A, Sultmann H, Poustka A, Vingron M. B Hildebrandt3 Heinrich Heine University, Du¨sseldorf, Germany Variance stabilization applied to microarray data calibration and B Royer-Pokora3 2 to the quantification of differential expression. Bioinformatics R Eils 2002; 18: 96–104. 1 N Gattermann 8 Goss Tusher V, Tibshirani R, Chu G. Significance analysis of 1 R Haas microarrays applied to the ionizing radiation response. Proc Natl R Kronenwett1 Acad Sci 2001; 98: 5116–5121.

Clonal aberrations in Philadelphia chromosome negative hematopoiesis in patients with chronic myeloid leukemia treated with imatinib or interferon alpha

Leukemia (2005) 19, 460–463. doi:10.1038/sj.leu.2403607 Between January 1997 and January 2004, we performed Published online 30 December 2004 cytogenetics and fluorescence in situ hybridization (FISH) analyses in 985 patients with CML. A total of 628 patients were treated with imatinib and 357 patients with interferon alpha TO THE EDITOR, (IFN). A minority of cases were treated in addition to IFN with The tyrosine kinase inhibitor imatinib mesylate was introduced hydroxyurea (HU), busulfan or thioguanin. All cases were in the treatment of chronic myeloid leukemia (CML) in 1998. In diagnosed and treated at our own institution or samples were 2002, there were first reports about the occurrence of clonal sent for diagnosis and follow-up studies to our reference À laboratory. In all, 128/985 patients were included in another changes in Philadelphia negative (Ph ) cells during imatinib 2 treatment. So far, more than 80 cases have been reported.1 report on clonal evolution during imatinib treatment. All 985 cases underwent routine cytogenetic analysis, interphase FISH and real-time PCR for BCR-ABL according to Correspondence: Dr U Bacher, Laboratory for Leukemia Diagnostics, standard protocols at diagnosis of CML.3 Complete cytogenetic Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, remission was defined as 0% Philadelphia-positive (Ph þ ) Marchioninistr. 15, D-81377 Munich, Germany; Fax: 49 89/7095 metaphases; major cytogenetic response: 1–34% Ph þ meta- 4971; E-mail: [email protected] þ Received 14 July 2004; accepted 19 October 2004; Published online phases; minor cytogenetic response: 35–95% Ph metaphases; þ 30 December 2004 no response: 96–100% Ph metaphases.

Leukemia Spotlight Correspondence 461 Table 1 Clinical data, cytogenetics and interphase FISH results

Number Gender Age Therapy Pretreatment Interval from Duration of Ph independent Ph+ positive Cytogenetic Cytomorphologyd of (years) diagnosis imatinib clonal clonal responsec patients (months)a treatment change evolution (months)b

1 F 53 Imatinib No 12 12 +8 +8 Minor CR 2 F 37 Imatinib No 17 11 +8 ins(11;11) Minor CR 3 F 61 Imatinib No 9 8 +8 No Minor - 4 F 31 Imatinib HU 6 3 +8 i(17q) Minor - 5 M 46 Imatinib IFN, HU 65 8 +8 +8 Minor CR 6 F 52 Imatinib No 10 7 +8 No Major CR 7 F 37 Imatinib IFN, HU 16 8 +8 No Minor - 8 F 29 Imatinib IFN, HU 32 14 +8 No Major - 9 F 59 Imatinib IFN 26 11 +8 +Phil Complete CR 10e F 32 Imatinib No 15 15 +8 No Complete - 11 F 45 Imatinib IFN 27 7 t(8;11) No Major - 12 F 69 Imatinib No 15 3 À7 No Complete CR 13 M 74 Imatinib HU, IFN, Ara C 69 5 À7 No Complete MDS/RAEB-2 14 F 67 Imatinib Busulfan, 35 3 À7 No Major MDS/RARS anagrelide, IFN, HU 15 M 77 Imatinib HU, IFN 37 4 del(7q) No Minor - 16 M 74 IFN No 18 6 +8 ÀY Minor CR HU: hydroxyurea; IFN: interferon; Ara C: cytarabin; CR: complete hematological remission of CML. aInterval from diagnosis of CML to the first occurrence of the Ph independent clonal changes. bInterval from start of imatinib/IFN therapy to the first occurrence of the Ph independent clonal changes. cCytogenetic response at the time point of the first occurrence of the Ph independent clonal changes. dCytomorphologic evaluation at the time point of the first occurrence of Ph independent clonal changes. ePhÀ BCR-ABL-positive CML.

100 80 60 40 20

% of metaphases 0 0 5 9 1317212631 months from start of imatinib Ph+ metaphases 8+ metaphases normal metaphases

Figure 1 Patient #3 was treated with imatinib as first-line therapy for CML in chronic phase with 100% Ph þ metaphases. Trisomy 8 in Ph independent cells was detected 8 months after the start of imatinib therapy in 8% of all metaphases. The proportion of Ph þ metaphases had decreased to 76%. At 19 months after the start of imatinib therapy, the patient had reached complete hematologic remission and major cytogenetic response with a reduction of the Ph þ metaphases to 10%. The proportion of Ph independent metaphases with trisomy 8 showed an increase to 90%. At 31 months after the start of imatinib therapy, the cytogenetic response to imatinib deteriorated with an increase of the Ph þ metaphases À from 10 to 36%. The Ph metaphases with trisomy 8 decreased from 90 to 36%. SPOTLIGHT

Philadelphia independent clonal evolution was found in 2.4% had been started in chronic phase. Patients’ characteristics are of all patients receiving imatinib (15/628) and in 0.2% of all demonstrated in Table 1. patients receiving IFN (1/357 patients). The incidence of Imatinib was first-line therapy in 40% (6/15). In total, 60% of Philadelphia independent clonal evolution during imatinib all imatinib patients (8/14) had received pretreatment with IFN, was in the range of the literature (2–17%).1,4,5 The lower hydroxyurea, busulfan, cytarabin or anagrelide prior to imatinib incidence of Philadelphia independent clonal evolution in therapy. In previous studies, the proportion of patients who had patients with interferon corresponds to the small number of received pretreatment was higher.1,4 anecdotal reports in the literature.1,6 The median interval from the start of therapy with imatinib Imatinib was started in 14/15 patients in chronic phase of or IFN to the first observation of the Ph independent CML. Accelerated phase was found in one case at the start of clonal aberrations was 7.5 months (range 3–15 months). This imatinib treatment. One patient demonstrated PhÀ BCR-ABL- corresponds to the literature, reporting median intervals positive CML (patient #10). Only one patient with new from 5 to 24 months.1,7,8 From diagnosis of CML to the first independent markers received IFN as first-line therapy, which occurrence of the Ph independent clonal changes, the median

Leukemia Spotlight Correspondence 462 100 80 60 40 20 % of metaphases 0 0 6 8 11162328 months from start of imatinib

Ph+ Ph-8 + Ph+8 + normal metaphases

Figure 2 Patient #5 was treated for CML in chronic phase for 23 months with IFN and for 5 months with hydroxyurea (HU). Owing to progression to accelerated phase, HU was stopped and imatinib was started. At that time Ph þ metaphases were 100%. The follow-up 6 months later showed Ph þ clonal evolution with evidence of trisomy 8. At 8 months after the start of imatinib therapy, the patient showed minor cytogenetic response with Ph þ metaphases decreasing to 60%. For the first time, a Ph independent clone with evidence of trisomy 8 was detected in 20% of all metaphases. Normal metaphases accounted for 8% only. At 28 months after the start of imatinib therapy, the patient reached þ

SPOTLIGHT complete cytogenetic remission of CML (with 0% of Ph metaphases). The percentage of Ph independent metaphases with trisomy 8 had increased continuously from 20 to 100%. Cytomorphologic examination and laboratory parameters revealed complete hematologic remission.

100 80 60 40 20 % of metaphases 0 1691216 months from start of imatinib Ph+ metaphases metaphases with double Ph chromosome Ph- +8 metaphases normal metaphases

Figure 3 Patient #9 was treated with IFN for CML in chronic phase. At 8 months after IFN had been started, major cytogenetic response was documented with a decrease of Ph þ metaphases from 96 to 12%. At this time point, clonal evolution in the Ph þ cells was detected with a doubling of the Ph chromosome. At 28 months after start of interferon, treatment was changed to imatinib because of relapse to chronic phase and development of refractoriness to interferon. At 8 months after change to imatinib, major cytogenetic response with a reduction of the Ph þ metaphases from 100 to 12% was documented. Cytomorphological examination demonstrated complete hematologic remission. At this time point, Ph independent cells with trisomy 8 were detected in 53% of all metaphases. Normal metaphases accounted for 35%. The follow-ups 12 and 16 months after the start of imatinib therapy showed a decrease of the PhÀ þ 8 clone from 53 to 16% and an increase of the normal metaphases from 35 to 72%. CML showed major cytogenetic response (with 12% Ph þ metaphases) and hematologic complete remission.

interval was 17.5 months (range 6–69 months). After the The patient with IFN treatment demonstrated trisomy 8 in PhÀ detection of the Philadelphia independent clone, we performed cells. Trisomy 8 in Ph independent cells in association to IFN in cytogenetic follow-up in our patients with a range of 0–27 CML was described before as the most frequent aberration.6 months. At the onset of the Ph independent clonal changes during In the 15 cases receiving imatinib, the most frequent treatment with imatinib or interferon, complete cytogenetic chromosomal change in PhÀ cells was trisomy 8 (67%; 10/15 remission was found in another 25% (4/16 patients) and major cases). Monosomy 7 was found in three cases (20%). One cytogenetic response in 25% (4/16). Minor cytogenetic remis- patient demonstrated del(7)(q11q22). A reciprocal translocation sion was observed in 50% (8/16). Our findings were in contrast t(8;11)(q22;q23) was found in another case. to the literature reporting that most patients with evidence of Ph However, Philadelphia independent clonal changes demon- independent clonal evolution were at complete cytogenetic strate a heterogenous spectrum. Trisomy 8 and monosomy 7 remission or showed major cytogenetic response.6–8 were found as the most frequent abnormalities.1,7 Reciprocal In all, 38% (6/16) of all patients demonstrated also clonal translocations were described in Philadelphia independent evolution with the Philadelphia translocation. A temporal clonal evolution in the literature.1,4,8 Reciprocal translocations coincidence of Ph þ and Ph independent clonal evolution at the involving 8q have not been reported so far in Philadelphia onset of the chromosomal changes in PhÀ cells was found in 31% independent clonal evolution. (5/16).

Leukemia Spotlight Correspondence 463 At the onset of the Philadelphia independent clonal changes, respect to the prognostic role of these specific abnormalities in 10/12 cases (83%) demonstrated complete hematologic remis- Ph independent clonal evolution. sion of CML at cytomorphologic examination of bone marrow Preliminary data suggest that Ph independent clonal evolution smears. Treatment-associated myelodysplastic syndrome (MDS) is associated with a higher risk of secondary MDS or AML. Thus, was observed in 2/16 patients (13%), both with evidence of Ph it is essential to identify cases with this specific risk profile in a independent monosomy 7 during imatinib. One patient clinical setting. This is only possible by continuation of demonstrated MDS with the subtype refractory anemia with cytogenetic follow-up even after complete or major cytogenetic ringsideroblasts (MDS/RARS) 3 months after the start of imatinib response to imatinib to identify all cases of Ph independent therapy in association to the primary detection of Ph indepen- cytogenetic aberrations and to achieve more understanding for dent monosomy 7 (#14). At this time point, the patient had the necessary therapeutical strategies in cases demonstrating this achieved major cytogenetic response of CML. This patient had a phenomenon. It is unclear whether patients showing Ph history of intensive pretreatment with IFN, busulfan, hydro- independent clonal changes should be taken off the study or xyurea, and anagrelide over 13 months. offered an allogeneic stem cell transplantation.4 In another patient (#13), we diagnosed MDS of the subtype U Bacher1 1Laboratory for Leukemia Diagnostics, refractory anemia with an excess of blasts (MDS/RAEB-2) with 2 15% bone marrow blasts and trilineage dysplasia 6 months after A Hochhaus Department for Internal Medicine III, Klinikum U Berger2 Grosshadern, Ludwig-Maximilians-University, the occurrence of the Ph independent monosomy 7. At this time, W Hiddemann1 Munich, Germany; and 2 CML was in complete cytogenetic remission. The patient was R Hehlmann2 III. Medizinische Klinik, Fakulta¨tfu¨r Klinische treated with imatinib for 11 months, following pretreatment with T Haferlach1 Medizin Mannheim der Universita¨t, Heidelberg, hydroxyurea, IFN, and cytarabin for another 64 months before. C Schoch1 Germany The pathogenesis of Ph independent evolution is still under discussion. Genomic instability in patients with CML was suggested.7 Further, a multistep model of the development of CML was discussed. This implicates that the Ph translocation References induces CML with the contribution of other clonal changes. Imatinib could select these pre-existing chromosomal changes.5 1 Terre C, Eclache V, Rousselot P, Imbert M, Charrin C, Gervais C et al. This hypothesis is underlined by the retrospective FISH analyses Report of 34 patients with clonal chromosomal abnormalities in 1 Philadelphia-negative cells during imatinib treatment of Philadel- performed by Terre et al who were able to detect the respective phia-positive chronic myeloid leukemia. Leukemia 2004; 18: 1340– Ph independent clonal aberration in 4/15 cases in samples that 1346. had been archived before the start of imatinib therapy. It is also 2 Schoch C, Haferlach T, Kern W, Schnittger S, Berger U, Hehlmann possible that imatinib may induce cytogenetic abnormalities. R et al. Occurrence of additional chromosome aberrations in It was suggested that Ph independent clonal evolution could chronic myeloid leukemia patients treated with imatinib mesylate. be induced by pretreatment or selected by imatinib.4 Leukemia 2003; 17: 461–463. 3 Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger The permanent inhibition of the ABL gene with its association U et al. Comparison of chromosome banding analysis, interphase- to proteins involved in DNA repair could lead to an accumula- and hypermetaphase-FISH, qualitative and quantitative PCR for tion of genetic damage.4 This pathogenesis can be discussed diagnosis and for follow-up in chronic myeloid leukemia: a study in some patients, but is not sufficient for explanation in all on 350 cases. Leukemia 2002; 16: 53–59. cases.8 4 Bumm T, Muller C, Al Ali HK, Krohn K, Shepherd P, Schmidt E À It has to be discussed further if Ph independent clonal et al. Emergence of clonal cytogenetic abnormalities in Ph cells in changed cells have a proliferation advantage in all cases. Bumm some CML patients in cytogenetic remission to imatinib but 4 et al observed in all patients an increase of the proportion of Ph restoration of polyclonal hematopoiesis in the majority. Blood independent cells with clonal changes during continuation of 2003; 101: 1941–1949. imatinib. 5 Meeus P, Demuynck H, Martiat P, Michaux L, Wouters E, We performed close cytogenetic follow-up in three cases after Hagemeijer A. Sustained, clonal karyotype abnormalities in the Ph independent clonal aberration had been detected. These Philadelphia chromosome negative cells of CML patients success- fully treated with imatinib. Leukemia 2003; 17: 465–467. three patients were treated with imatinib and all demonstrated 6 Ariyama T, Inazawa J, Uemura Y, Kakazu N, Maekawa T, Urase F trisomy 8. In contrast to this observation, Ph independent clonal et al. Clonal origin of Philadelphia-chromosome negative cells changed metaphases, normal metaphases, and Ph þ metaphases with trisomy-8 appearing during the course of alpha-interferon SPOTLIGHT did not follow a recurrent pattern in three of our patients with therapy for Ph positive chronic myelocytic-leukemia. Cancer trisomy 8 (see Figures 1–3). Genet Cytogenet 1995; 81: 20–23. None of the 11 patients with trisomy 8 during imatinib or 7 Medina J, Kantarjian H, Talpaz M, O’Brien S, Garcia-Manero G, Giles F et al. Chromosomal abnormalities in Philadelphia interferon treatment showed cytomorphological features of MDS chromosome-negative metaphases appearing during imatinib or AML. In contrast, we observed treatment-associated MDS in mesylate therapy in patients with Philadelphia chromosome- two of four cases showing Ph independent monosomy 7 or positive deletion of 7q during imatinib treatment. chronic myelogenous leukemia in chronic phase. Cancer 2003; Some more cases of MDS and of transformation to AML were 98: 1905–1911. reported in association to monosomy 7 during imatinib 8 O’Dwyer ME, Gatter KM, Loriaux M, Druker BJ, Olson SB, 4 Magenis RE et al. Demonstration of Philadelphia chromosome treatment of CML. Monosomy 7 is associated with an inferior negative abnormal clones in patients with chronic myelogenous prognosis in MDS and AML. However, the limited follow-up leukemia during major cytogenetic responses induced by imatinib and the limited samples do not allow definite conclusions with mesylate. Leukemia 2003; 17: 481–487.

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