High Expression Levels of X-Linked Inhibitor of Apoptosis Protein and Survivin Correlate with Poor Overall Survival in Childhood De Novo Acute Myeloid Leukemia

Vol. 10, 3737–3744, June 1, 2004 Clinical Cancer Research 3737

High Expression Levels of X-Linked Inhibitor of Apoptosis Protein and Survivin Correlate with Poor Overall Survival in Childhood de Novo Acute Myeloid Leukemia

Ingo Tamm,4 Stephan Richter,1 a mean follow-up of 34 months, a shorter overall survival ؍ Doreen Oltersdorf,1 Ursula Creutzig,2 was associated with high expression levels of XIAP [30 (n P < 0.05] and survivin [27 ;(34 ؍ versus 41 months (n (10 4 3 ؍ ؍ ,Jochen Harbott, Frank Scholz 1 1 (n 10) versus 41 months (n 34); P < 0.05]. Leonid Karawajew, Wolf-Dieter Ludwig, and Conclusions: We conclude that apoptosis-related mole- 1 Christian Wuchter cules are associated with maturation stage, cytogenetic risk 1HELIOS Klinikum Berlin, Robert-Ro¨ssle-Klinik, Department of groups, and therapy outcome in childhood de novo AML. Hematology, Oncology, and Tumor Immunology, Charite´, Campus 2 The observed association of XIAP with immature FAB Berlin-Buch, Universita¨tsmedizin Berlin, Berlin; Department of types, intermediate/poor cytogenetics, and poor overall sur- Pediatric Hematology/Oncology, University Children’s Hospital Mu¨nster, Munster; 3Oncogenetic Laboratory, Children’s Hospital, vival should be confirmed within prospective pediatric AML Justus-Liebig-University of Giessen, Giessen; and 4Department of trials. Hematology and Oncology, Charite´, Campus Virchow, Humboldt- University of Berlin, Berlin, Germany INTRODUCTION During the last years, it became evident that apoptosis- ABSTRACT related molecules may be useful as prognostic markers because Purpose: Apoptosis-related proteins are important mol- several antiapoptotic mechanisms are operational in acute leu- ecules for predicting chemotherapy response and prognosis kemias. Suppression of apoptosis contributes to leukemogenesis in adult acute myeloid leukemia (AML). However, data on by different mechanisms, including prolonging cell life span, the expression and prognostic impact of these molecules in thus facilitating the accumulation of gene mutations, permitting childhood AML are rare. growth factor-independent cell survival, promoting resistance to Experimental Design: Using flow cytometry and West- immune-based cytotoxicity, and allowing disobeyance of cell ern blot analysis, we, therefore, investigated 45 leukemic cell cycle checkpoints which would normally induce apoptosis. De- samples from children with de novo AML enrolled and fects in apoptotic mechanisms also play an important role in treated within the German AML-BFM93 study for the ex- resistance to chemotherapy (1–3). In adult acute myeloid leu- pression of apoptosis-regulating proteins [CD95, Bcl-2, Bax, kemia (AML), the expression patterns and prognostic role of Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis pro- CD95 (Fas/APO-1), molecules of the Bcl-2 and caspase families tein (XIAP), cellular inhibitor of apoptosis protein–1 (cIAP- as well as members of the inhibitors of apoptosis proteins (IAPs) 1), survivin]. have been investigated in a variety of studies (4–15). In con- Results: XIAP (P < 0.002) but no other apoptosis reg- trast, systematic data on the expression and prognostic impact of ulators showed maturation-dependent expression differ- these molecules in pediatric AML are rare. ences as determined by French-American-British (FAB) Childhood AML has a better treatment outcome than adult morphology with the highest expression levels observed AML. For the current chemotherapy regimens, these prognostic within the immature M0/1 subtypes. XIAP (P < 0.01) and differences are true for response to induction chemotherapy, Bcl-xL (P < 0.01) expression was lower in patients with relapse frequency, and overall survival (16–19). It has been favorable rather than intermediate/poor cytogenetics. After speculated that differences in the maturation stage of the transformed progenitor cell clone contribute to the distinct prognosis of age-related AML groups. The leukemic cells in adult AML patients may arise from a transformed undifferentiated hematopoietic stem cell, whereas in childhood AML, the malignant cell Received 11/25/03; revised 2/10/04; accepted 2/16/04. Grant support: This study was supported by grants from the Carreras- clone potentially arises from a more committed myeloid pro- Foundation, Mildred-Scheel-Stiftung fu¨r Krebsforschung, and Deutsche genitor cell. Different maturation stages of hematopoietic pro- Forschungsgemeinschaft. genitor cells differ in their intrinsic resistance toward chemo- The costs of publication of this article were defrayed in part by the therapy-induced cell death (i.e., immature stem cells are more payment of page charges. This article must therefore be hereby marked resistant than committed myeloid progenitor cells), and this fact advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. may contribute to the different treatment outcome of age-related Note: C. Wuchter is currently at PAREXEL Medical Services Europe, AML groups (20, 21). Thus, age-related acute leukemia groups Klinikum Westend, Haus 18, Spandauer Damm 130, 14050 Berlin, might be associated with distinct expression patterns of apopto- Germany. sis-related molecules leading to the distinct prognosis in adult Requests for reprints: Ingo Tamm, Department of Hematology and Oncology, Charite´, Campus Virchow, Universita¨tsmedizin Berlin; Au- and childhood acute leukemia (11, 22, 23). gustenburger Platz 1, 13353 Berlin, Germany. Phone: 49-30-450-559- The main aims of this study were to analyze the expression 438; Fax: 49-30-450-559-958; E-mail: [email protected]. patterns and prognostic relevance of apoptosis-related proteins

Table 1 Characteristics of 45 examined children with de novo acute myeloid leukemia (AML) treated according to the AML-BFM93 protocol No. of patients 45 (21 males, 24 females) Age in years, mean (range) 9 (0–16) Initial hemoglobin g/dl, mean (range) 8.3 (3.7–14.4) Initial WBC ϫ 109/liter, mean (range) 71.739 (1.500–355.500) Initial platelet count ϫ 109/liter, mean (range) 61.104 (5.000–343.000) Initial LDHa units/liter, mean (range) 784 (192–2.584) FAB morphology M0 (n ϭ 3); M1 (n ϭ 7); M2 (n ϭ 9); M3 (n ϭ 5); M4 (n ϭ 11); M5 (n ϭ 6); M6 (n ϭ 1) Cytogenetics risk groups Good (n ϭ 7); intermediate/poor (n ϭ 30) a LDH, lactate dehydrogenase; FAB, French-American-British (morphology types).

in childhood de novo AML; to correlate expression with karyo- mg/m2, days 4 and 5) either before or after standard consolida- type, maturation stage (FAB subtypes, immunophenotype), and tion therapy. Randomized scheduling of HAM after induction outcome (response to induction chemotherapy, relapse fre- did not reveal major differences in outcome. However, high-risk quency, overall survival) of pediatric AML cases who entered patients with ADE (ara-C, daunorubicin, etoposide) as induction the AML-BFM93 study and, thus, received standardized diag- therapy benefited especially from early HAM (18). After con- nostic work-up and therapy, and to assess the applicability of solidation therapy, all of the patients were treated with an these proteins as markers for treatment response. intensification block of high-dose ara-C and VP-16 (high-dose ara-C 3 g/m2 twice daily for 3 days and etoposide 125 mg/m2, MATERIALS AND METHODS day 2 to 5). This was followed by cranial irradiation with 18 Gy Patient Samples. Patient-derived cell samples (n ϭ 45; (standard dose in children Ն3 years) and maintenance therapy bone marrow n ϭ 35, peripheral blood n ϭ 10) were consecu- of thioguanine 40 mg/m2 p.o. and ara-C 40 mg/m2 s.c. for 4 days tively collected, ficoll-purified, and cryopreserved between monthly for a total of 18 months. 1997 and 1999 within the German multicenter AML-BFM93 Of the 45 patients in this series, 35 patients were treated study. Diagnosis of AML was made morphologically based on according to high-risk therapy regimen and 10 patients received FAB criteria (24, 25) and immunophenotypically according to standard-risk treatment. Three patients died during induction European Group for the Immunological Characterization of chemotherapy, 3 patients were nonresponders, and 39 patients Leukemias (EGIL) recommendations (26). All of the samples achieved complete remission (CR). Of the 39 patients with CR, contained more than 80% leukemic cells based on morpholog- 27 patients are in continuous CR (mean event-free survival: 42 ical and immunophenotypical criteria. All of the patients were months) and 12 patients suffered from relapse (mean event-free- children and had de novo AML. Clinical characteristics of these survival: 10 months). The mean overall survival follow up is 34 patients are summarized in Table 1. months. Treatment According to German AML-BFM93 Study Expression Analysis of Apoptosis-Related Molecules Protocol. Treatment modalities and risk stratification of the CD95, Bcl-2, and Bax by Flow Cytometry. Surface CD95 AML-BFM93 study are described in detail elsewhere (18). In expression and intracellular expression of Bcl-2 and Bax were brief, during induction phase, patients received either ADE determined as described previously using the phycoerythrin- (ara-C: 100 mg/m2, day 1 to 8; daunorubicin 30 mg/m2 twice conjugated anti-CD95 monoclonal antibody DX2, the FITC- daily, days 3 to 5; etoposide 150 mg/m2,day6to8)orAIE conjugated anti-Bcl-2 monoclonal antibody 124, and the poly- (idarubicin 12 mg/m2 daily instead of daunorubicin). Although clonal rabbit-antihuman antibody I-19 raised against Bax- AIE was associated with a better blast reduction in the bone specific peptide sequences (11). Nonspecific binding due to marrow on day 15, long-term therapy outcome for both induc- Fc-receptors was blocked by preincubation of the cells with tion regimens was similar (27). After induction, patients were polyclonal rabbit serum (Life Technologies, Inc., Paisley, treated according to risk level (standard-risk group: FAB M1 or United Kingdom). Antigen expression distribution in individual M2 with Auer rods, FAB M3, and FAB M4Eo with Յ5% blasts cell samples was quantified as relative fluorescence intensity, in the bone marrow on day 15; high-risk group: all others). determined by the ratio of mean fluorescence intensity of cells Standard-risk patients were shifted to the high-risk group if they stained for the respective antigen to mean fluorescence intensity had more than 5% blasts in the bone marrow on day 15. Patients of the corresponding negative control. Bax:Bcl-2 ratio was with FAB M3 were always treated within standard-risk group, determined by dividing the Bax relative fluorescence intensity regardless of blast count on day 15. All of the patients received value by the Bcl-2 relative fluorescence intensity value. 6 weeks of standard consolidation therapy with seven drugs Western Blot Analysis of Bcl-xL, Procaspase-3, XIAP, (thioguanine 60 mg/m2, day 1 to 43; prednisolone 40 mg/m2, cIAP-1, and Survivin. Immunoblotting was carried out using day 1 to 28; vincristine 1.5 mg/m2 and doxorubicin 30 mg/m2, cell lysates from mononuclear cell fractions generated by ficoll day 1, 8, 15, an 22; ara-C 75 mg/m2 day 3 to 6, 10 to 13, 17 to separation. Detergent-lysates were prepared in the presence of 20, 24 to 27, 31 to 34, and 38 to 41; intrathecal ara-C 40 mg in protease inhibitors as described previously (8, 28). After nor- children Ն3 years, day 1, 15, 29, and 43; cyclophosphamide 500 malization for total protein content (20 ␮g/Lane), samples were mg/m2 day 29 and 43). High-risk patients were randomized to subjected to SDS-PAGE/immunoblot analysis using mono- receive additional consolidation treatment with HAM (high- clonal antibodies specific for X-linked inhibitor of apoptosis dose ara-C 3 g/m2 twice daily, days 1–3; mitoxantrone 10 protein (XIAP; Transduction Laboratories, Lexington, KY), cel-

Table 2 Correlation of expression of apoptosis-related proteins with French-American-British (FAB) morphology (M0/1, M2/3, M4/5) in childhood de novo acute myeloid leukemia CD95, Bax, and Bcl-2 mean expression levels are given as relative fluorescence intensity values, and Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis protein (XIAP), survivin, and cellular inhibitor of apoptosis–1 (cIAP-1) expression levels are given as standardized mean values (relative absorbance) as described in “Materials and Methods.” Expression levels CD95 Bax Bcl-2 Bcl-xL Procaspase-3 XIAP Survivin cIAP-1 M0/1 Mean 4.2 4.4 2.7 3606 5600 5449 207 8354 na 10 10 10 9 9 9 9 8 SE 1.0 0.7 0.4 1175 1187 1060 143 1631 M2/3 Mean 3.6 5.4 2.6 1253 2674 678 246 6577 n 11 11 11 14 14 14 14 3 SE 0.4 0.8 0.3 482 560 342 177 1624 M4/5 Mean 3.3 5.4 2.5 1874 3868 2810 500 8216 n 14 14 14 17 17 17 17 11 SE 0.6 0.5 0.3 350 605 737 228 2081 P ns ns ns ns ns 0.002 ns ns a n, number of examined samples; ns, not significant.

lular inhibitor of apoptosis protein–1 (cIAP-1), survivin (R&D RESULTS Systems, Minneapolis, MN), procaspase-3 (PharMingen, San Constitutive Expression of Apoptosis-Related Proteins Diego, CA), and Bcl-xL (Santa Cruz Biotechnology, Santa in Childhood de Novo AML: Correlation with FAB Mor- Cruz, CA). Data on X-ray films were quantified by scanning- phology and Immunophenotype. Morphological FAB data densitometry using the NIH Image analysis system. To normal- were grouped into an immature subgroup (M0/1) and subtypes ize for variation in antibody concentration or time of exposure, with granulocytic (M2/3) and myelomonocytic (M4/5) differen- the protein signal from the respective patient was normalized tiation (Table 2). Immunophenotypic data were divided into against the protein signal of the control cell line BJAB (human immature phenotype (CD34, CD117 positivity) and phenotypic Burkitt-like lymphoma cell line). Western blot results are ex- features of granulomonocytic (CD4, CD14, CD15, CD36, pressed in terms of this ratio (relative absorbance). CD64, CD65, HLA-DR positivity) differentiation (Table 3). Cytogenetics. Cytogenetic analyses were performed ac- Within this study, expression of CD95 was examined in 39 cording to standard protocols. Cytogenetic data were classified cell samples (Fig. 1B). In adult de novo AML, CD95 expression according to the International System for Human Cytogenetic is higher in mature than in immature FAB subtypes (10, 11, 13). Nomenclature (ISCN; Ref. 29) and were grouped into favorable In contrast, expression of CD95 showed no clear maturation- [t(8;21), inv(16)/t(16;16), t(15;17)] and intermediate/poor risk dependent differences in this series of pediatric AML cases categories [normal karyotype, other abnormalities, Ϫ5/5q-, Ϫ7/ (Table 2). Expression of Bcl-2 was investigated in 38 cell 7q-, 11q23-abnormalities, inv(3)/t(3;3), t(9;;22), t(6;9), 17p- samples (Fig. 1D). Bcl-2 expression is associated with immature abnormalities, complex aberrant karyotypes] (30). FAB subtypes and CD34 expression in adult de novo AML (4, Statistical Analysis. Differences in the expression levels 11, 14, 15). Within this series of childhood AML samples, of apoptosis-related molecules between CD marker positive and expression of Bcl-2 did not differ significantly between FAB negative AML groups (e.g., CD34 positive versus CD34 nega- subtypes and CD34 positive and negative cases (Tables 2 and 3). tive), FAB subtypes, and cytogenetic risk groups were evaluated XIAP expression levels were examined in 44 cell samples using either Mann-Whitney or Kruskal-Wallis tests. The expres- and were significantly higher in immature compared with more sion levels of apoptosis-related molecules were correlated with mature FAB subtypes (Table 2, Fig. 1A). For XIAP, this is in clinical data (age, WBC, platelet count, hemoglobin value, contrast to adult de novo AML; high expression of XIAP was lactate dehydrogenase) in individual cell samples by bivariate associated with myelomonocytic FAB subtypes M4/5 and a monocytic-differentiated immunophenotype in a study of adult correlation (Spearman correlation coefficient r ). Differences in s de novo AML patients (positivity for CD4, CD14, CD36, and expression levels of these molecules between patients with HLA-DR).5 Similar to previously observed expression patterns complete response after induction chemotherapy and nonre- in adult AML (11), expression of Bax (Fig. 1C), survivin, and sponders, as well as between patients with a relapse and patients cIAP-1 proteins (investigated in 38, 44, and 23 cell samples, still in complete remission, were compared using ANOVA test. respectively; Fig. 1A) showed no clear-cut correlations with Overall survival was calculated from the date of diagnosis to death from any cause or last follow-up, and respective survival curves were obtained according to Kaplan-Meier (log rank test). Ps Ͻ 0.05 were considered statistically significant. All of the statistical analyses were done with the SPSS software program. 5 Unpublished data.

Table 3 Expression of apoptosis-related proteins in childhood de novo acute myeloid leukemia: Correlation with immunophenotype Bcl-2 mean expression levels are given as relative fluorescence intensity (RFI) values and Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis protein (XIAP), and survivin expression levels are given as standardized mean values relative absorbance (ROD) as described in “Materials and Methods.” Cell samples were considered positive for a specific CD marker if the antigen was expressed on at least 20% of the leukemic cells. CD95, Bax, and cellular inhibitor of apoptosis protein–1 (cIAP-1) expression levels did not correlate with any of the listed CD markers, and the respective data are, therefore, not included in this Table. Bcl-2 Bcl-xL Procaspase-3 XIAP Survivin CD markers RFI Ϯ SE na P ROD Ϯ SE nPROD Ϯ SE nPROD Ϯ SE nPROD Ϯ SE nP CD34 pos. 2.6 Ϯ 0.2 20 ns 1644 Ϯ 284 24 ns 4110 Ϯ 515 24 ns 1565 Ϯ 455 24 ns 368 Ϯ 143 24 ns CD34 neg. 2.6 Ϯ 0.3 17 2568 Ϯ 683 19 3449 Ϯ 715 19 3709 Ϯ 814 19 410 Ϯ 195 19 CD117 pos. 2.7 Ϯ 0.2 30 ns 2183 Ϯ 415 33 ns 4205 Ϯ 497 33 ns 2340 Ϯ 508 33 ns 234 Ϯ 92 21 ns CD117 neg. 2.1 Ϯ 0.2 7 1621 Ϯ 564 10 2539 Ϯ 696 10 3082 Ϯ 1120 10 889 Ϯ 367 10 CD4 pos. 2.5 Ϯ 0.2 14 ns 2551 Ϯ 622 17 ns 4682 Ϯ 777 17 ns 3745 Ϯ 880 17 0.04 634 Ϯ 248 17 ns CD4 neg. 2.7 Ϯ 0.3 23 1726 Ϯ 394 26 3253 Ϯ 465 26 1706 Ϯ 457 26 225 Ϯ 95 26 CD14 pos. 1.7 Ϯ 0.1 7 0.01 1671 Ϯ 492 10 ns 2742 Ϯ 760 10 ns 1631 Ϯ 708 10 ns 655 Ϯ 337 10 ns CD14 neg. 2.8 Ϯ 0.2 30 2168 Ϯ 424 33 4144 Ϯ 495 33 2780 Ϯ 563 33 305 Ϯ 112 33 CD15 pos. 2.5 Ϯ 0.2 21 ns 1427 Ϯ 266 24 ns 3033 Ϯ 523 24 ns 2124 Ϯ 532 24 ns 436 Ϯ 168 24 ns CD15 neg. 2.7 Ϯ 0.3 16 2842 Ϯ 669 17 4809 Ϯ 643 19 3003 Ϯ 811 19 325 Ϯ 159 19 CD36 pos. 1.9 Ϯ 0.1 10 0.02 1833 Ϯ 394 13 ns 3701 Ϯ 715 13 ns 2996 Ϯ 962 13 ns 840 Ϯ 290 13 0.019 CD36 neg. 3.0 Ϯ 0.2 25 2186 Ϯ 491 28 3856 Ϯ 531 28 2390 Ϯ 558 28 204 Ϯ 98 28 CD64 pos. 2.4 Ϯ 0.2 26 ns 1676 Ϯ 291 30 ns 3559 Ϯ 452 30 ns 2265 Ϯ 547 30 ns 408 Ϯ 154 30 ns CD64 neg. 3.0 Ϯ 0.4 11 2921 Ϯ 896 13 4414 Ϯ 951 13 3084 Ϯ 888 13 338 Ϯ 151 13 CD65 pos. 2.5 Ϯ 0.2 25 ns 1671 Ϯ 290 31 ns 3223 Ϯ 447 31 0.04 2324 Ϯ 542 31 ns 498 Ϯ 153 31 ns CD65 neg. 2.7 Ϯ 0.4 12 3037 Ϯ 948 12 5354 Ϯ 873 12 3000 Ϯ 920 12 98 Ϯ 98 12 HLA-DR pos. 2.5 Ϯ 0.2 30 ns 2471 Ϯ 424 32 0.03 4446 Ϯ 489 32 0.01 3033 Ϯ 564 32 ns 390 Ϯ 131 32 ns HLA-DR neg. 2.9 Ϯ 0.3 7 833 Ϯ 339 11 1992 Ϯ 595 11 999 Ϯ 605 11 377 Ϯ 255 11 a n, number of examined samples; pos., positive; neg., negative; ns, not significant.

FAB subtypes (Table 2) and immunophenotype (data not risk therapy protocol and patients who received standard-risk shown) in this series of pediatric AML cases. treatment. Constitutive Expression of Apoptosis-Related Proteins Three patients of the 45 examined patients were nonre- in Childhood de Novo AML: Correlation with Cytogenetic sponders to induction chemotherapy and 3 patients died during Risk Groups. Cytogenetic analyses were available for 37 this therapy phase (“early deaths”). We observed no clear-cut patients and grouped into favorable and intermediate/poor risk expression differences for any of the analyzed proteins between categories as described in “Materials and Methods.” Interest- nonresponders and patients with CR (n ϭ 39) within this study ingly, all of the cell samples of the seven patients with favorable (data not shown). Moreover, a significant expression difference cytogenetics were negative for XIAP and Bcl-xL. In contrast, between relapsed patients (n ϭ 12) and patients in continuous XIAP and Bcl-xL were detectable in all of the cell samples of CR (n ϭ 27) was not seen for any of the investigated apopto- the 30 patients with intermediate/poor risk cytogenetics (XIAP: sis-related molecules in this series (data not shown). 2509 Ϯ 503 relative absorbance, P Ͻ 0.01; Bcl-xL: 2615 Ϯ 439 However, fitting with expectations based on the antiapop- relative absorbance, P Ͻ 0.01). Expression levels of none of the totic activity of XIAP and survivin in cells, patients expressing other analyzed proteins correlated with cytogenetic risk groups high levels of XIAP (upper third expression level) had a shorter (data not shown). overall survival than patients expressing low levels of XIAP Constitutive Expression of Apoptosis-Related Proteins [mean, 30 (n ϭ 10) versus 41 months (n ϭ 34); P Ͻ 0.05; Fig. in Childhood de Novo AML: Correlation with Clinical Data 2A]. When patient groups with XIAP expression levels above and Therapy Response. Procaspase-3 expression levels were and below mean or median were compared, no significant ϭ ϭ Ͻ positively correlated with age (rs 0.3, n 44, P 0.05) and differences in overall survival were observed for these groups ϭ ϭ Ͻ WBC (rs 0.4, n 44, P 0.01). XIAP expression levels (data not shown). Patients with detectable survivin protein ex- ϭ ϭ Ͻ correlated with platelet count (rs 0.3, n 44, P 0.05). No pression had a shorter overall survival than patients negative for additional significant correlations between apoptosis-related survivin [mean 27 (n ϭ 10) versus 41 months (n ϭ 34); P Ͻ molecules and clinical data (age, WBC, platelet count, hemo- 0.05; Fig. 2B]. In contrast, none of the other investigated pro- globin value, lactate dehydrogenase) were found. With the ex- teins correlated with overall survival (data not shown). A recent ception of XIAP (standard risk: mean relative absorbance ϭ study indicates a high Bax:Bcl-2 ratio as a favorable prognostic 780, n ϭ 10; high risk: mean relative absorbance ϭ 2948, n ϭ marker for response to induction therapy and overall survival in 34; P Ͻ 0.05), expression levels of analyzed proteins did not adult de novo AML (4). In contrast, in this study, children (n ϭ differ significantly between patients treated according to high- 19) with a ratio above average had a shorter overall survival in

Fig. 1 Expression levels of apoptosis-related proteins in de novo acute myeloid leukemia (AML) cells. Examples of immunoblot (A) and flow cytometry (B–D) data are shown for three representative samples [French-American-British (FAB) morphology types M1, M3, M4). A, immunoblotting was carried out using cell lysates from mononuclear cell fractions generated by ficoll separation. Detergent-lysates were prepared in the presence of protease inhibitors. After normalization for total protein content (20 ␮g/Lane), samples were subjected to SDS-PAGE/immunoblot analysis using monoclonal antibodies specific for X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein–1 (cIAP-1), survivin, procaspase-3, and Bcl-xL. Data on X-ray films were quantified by scanning-densitometry using the NIH Image analysis system. To normalize for variation in antibody concentration or time of exposure, the protein signal from the respective patient was normalized against the protein signal of the control cell line BJAB ( B,human Burkitt-like lymphoma cell line). Expression levels are expressed in terms of this ratio (relative absorbance). Surface CD95 expression (B) and intracellular expression of Bax (C) and Bcl-2 (D) were determined using the phycoerythrin-conjugated anti-CD95 monoclonal antibody DX2, the FITC-conjugated anti-Bcl-2 monoclonal antibody 124, and the polyclonal rabbit-antihuman antibody I-19 raised against Bax-specific peptide sequences. Nonspecific binding due to Fc receptors was blocked by preincubation of the cells with polyclonal rabbit serum (Life Technologies, Inc.). Antigen expression distribution in individual cell samples was quantified as relative fluorescence (FL) intensity (RFI), determined by the ratio of mean fluorescence intensity of cells stained for the respective antigen to mean fluorescence intensity of the corresponding negative control.

the maturation stage of the transformed progenitor cell clone, including distinct expression patterns of apoptosis-related molecules, might contribute to the distinct prognosis of age-related AML groups (20, 21, 23). Previously, we showed that the overall expression levels of certain apoptosis-related molecules (cIAP-1, Bax, procaspase-3, XIAP) differ between childhood and adult AML (22). In this study, we provide further evidence that expression patterns of pro- and antiapoptotic molecules vary between these age groups. The previously described association of CD95 (10, 11, 13) and Bcl-2 (4, 14, 15, 22) expression with certain maturation stages of the leukemic cells in adult AML was not seen in this pediatric AML series. This finding indicates that gene transcription and protein expression of these molecules may be differently regulated within distinct age- related AML groups. Comparable with our previous findings in adult AML and normal peripheral blood cells,5 XIAP expression was higher in myelomonocytic-differentiated AML cells than in AML cells with granulocytic differentiation. Thus, the results of this study provide further evidence that XIAP plays a role in malignant as well as in normal monocytic differentiation. This assumption is also supported by the finding that XIAP is linked to monocytic differentiation in bryostatin1-treated leukemia cell lines in vitro and that down-regulation of XIAP blocks monocytic differentiation (31). Moreover, a recent study showed that induction of monocytic differentiation in the HL-60 cell line model leads to a marked up-regulation of XIAP, whereas during granulocytic differentiation, XIAP levels progressively declined (32). It will be interesting to study the molecular mechanisms for the differential expression of IAPs in AML cells in more detail. For example, recent data suggest that XIAP may be regulated by the phosphatidylinositol 3Ј-kinase pathway in AML (33). Cytogenetics are currently the most clearly defined prognostic factors in AML. Recent studies revealed important molecular insights in the transformational potential of specific leukemia-associated chromosomal abnormalities (34). Still, it is mostly unclear why genetic subgroups differ in their chemotherapy response and whether this is related to differences in cellular drug resistance (35). Fitting with the antiapoptotic activity of Fig. 2 Effect of apoptosis-related protein expression on overall sur- XIAP and Bcl-xL, the observed lower expression of these mol- vival in childhood de novo acute myeloid leukemia (AML). A, Kaplan- ecules in the favorable cytogenetic risk group might contribute Meier overall survival curves for AML patients stratified according to to the better prognosis of this risk group. This significant lower X-linked inhibitor of apoptosis protein (XIAP) protein levels (HIGH, upper one-third expression level; LOW, lowest two-thirds expression XIAP expression within the favorable cytogenetic risk group 5 levels). B, Kaplan-Meier overall survival curves for AML patients was also observed in our previous study in adult de novo AML. stratified according to survivin protein levels (HIGH, samples with Despite great advances in supportive care, chemotherapy is detectable survivin expression; LOW, survivin-negative samples). still associated with a significant amount of toxicity. The AML- BFM group currently investigates whether standard-risk patients in addition to the high-risk group might also benefit from an intensified consolidation therapy with HAM or whether this ϭ this series compared with children (n 19) with a ratio below approach will lead to a significant increase of toxic side-effects ϭ average (mean, 33 versus 40 months; P 0.175). outweighing the anticipated survival benefit (18). Thus, additional potential prognostic markers to define subgroups that DISCUSSION might benefit from specifically tailored therapy approaches are To determine the expression pattern and prognostic signif- urgently needed. The newly described association between high icance of apoptosis-related molecules in childhood de novo expression levels of the antiapoptotic proteins XIAP and sur- AML, we investigated 45 cell samples of children enrolled and vivin and a shorter overall survival in this series defines these treated within the German AML-BFM93 trial. molecules as potential prognostic markers in childhood AML. Thus far, it is unclear why childhood AML has a better For XIAP, this is in line with our previous finding suggesting treatment outcome compared with adult AML. Differences in this molecule as a potential prognostic marker in adult AML (8).

In contrast, we could not determine a prognostic role for sur- 7. Svingen PA, Karp JE, Krajewski S, et al. Evaluation of Apaf-1 and vivin in adult AML.5 Survivin is highly expressed and is cyto- procaspases-2, -3, -7, -8, and -9 as potential prognostic markers in acute kine regulated in myeloid leukemias (5) and is a negative leukemia. Blood 2000;96:3922–31. prognostic marker in a variety of solid tumors and diffuse large 8. Tamm I, Kornblau SM, Segall H, et al. Expression and prognostic significance of IAP-family genes in human cancers and myeloid leuke- B-cell lymphomas (1, 36). For example, it has been reported that mias. Clin Cancer Res 2000;6:1796–803. survivin expression in neuroblastomas correlates with clinically 9. Adida C, Recher C, Raffoux E, et al. Expression and prognostic more aggressive, histologically unfavorable disease (37). Higher significance of survivin in de novo acute myeloid leukaemia. Br J levels of survivin protein and p53 accumulation (indicative of Haematol 2000;111:196–203. mutant p53) were positively correlated in a survey of gastric 10. Lewis NR, Pallis M, Russell NH. Fas receptor-Fas ligand system is cancers, implying an association of survivin with more aggres- independent of both CD34 status and chemosensitivity in acute myeloid sive disease (38). Adida et al. (9) found no significant difference leukemia. Exp Hematol 2000;28:535–42. in remission rate or survival in adult AML patients expressing 11. Wuchter C, Karawajew L, Ruppert V, et al. Clinical significance high versus low levels of survivin. However, survivin expres- of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, sion became an independent negative prognostic factor for sur- maturation stage, and cytogenetics. Leukemia (Baltimore) 1999;13: vival when adjusted for established prognostic factors (cytoge- 1943–53. netics, age, and WBC). 12. Estrov Z, Thall PF, Talpaz M, et al. Caspase 2 and caspase 3 protein At least two limitations of this study must be kept in mind levels as predictors of survival in acute myelogenous leukemia. Blood for any further interpretation of the obtained results: (a) cryo- 1998;92:3090–7. preserved cell samples were used for our investigations. There- 13. Iijima N, Miyamura K, Itou T, Tanimoto M, Sobue R, Saito H. fore, this series might include a selection bias toward samples Functional expression of Fas (CD95) in acute myeloid leukemia cells in from patients with high leukocyte counts. The observed average the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy. Blood 1997;90:4901–9. leukocyte count within this series is indeed about 3-fold higher 14. Lauria F, Raspadori D, Rondelli D, et al. 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Ingo Tamm, Stephan Richter, Doreen Oltersdorf, et al.

Clin Cancer Res 2004;10:3737-3744.

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