Risk Assessment in Patients with Acute Myeloid Leukemia and a Normal Karyotype

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1 1416 Vol. 11, , February 15, 2005 Clinical Cancer Research Risk Assessment in Patients with Acute Myeloid Leukemia and a Normal Karyotype Marianne Bienz, Madleina Ludwig, Beatrice U. Mueller, Elisabeth Oppliger Leibundgut, Daniel Ratschiller, Max Solenthaler, Martin F. Fey, and Thomas Pabst Institute of Medical Oncology and the Laboratory for Molecular Diagnostics, Department of Hematology, University of Berne, Berne, Switzerland ABSTRACT Purpose: The recognition of a number of leukemiaspecific cytogenetic abnormalities and their role as independent prognostic factors have provided considerable insights into leukemia pathogenesis and have paved the way to adopt risk-adapted treatment. However, f50% of newly diagnosed acute myeloid leukemia (AML) have a normal karyotype. There has therefore been much interest in identifying molecular markers that could help to improve the prognostic stratification of patients with normal-karyotype AML. Experimental Design: Consecutive untreated AML patients (n = 67) from a single institution all with normal karyotype were analyzed for the presence of mutations in the myeloid transcription factor gene CEBPA (for CCAAT/ enhancer binding protein-a), for internal tandem duplications (ITD) of the tyrosine kinase receptor gene FLT3 (for fms-like tyrosine kinase 3), and for expression of the BAALC gene (for brain and acute leukemia, cytoplasmic). Results: 17.9% of normal-karyotype AML had mutations in the CEBPA gene, and 28.4% had FLT3-ITD; 65.7% (44 of 67) had high BAALC expression and 34.3% (23 of 67) had low BAALC expression. Patients with CEBPA mutations had a very favorable course of their disease. Median diseasefree survival (DFS) and overall survival (OS) were 33.5 and 45.5 months, respectively, compared with 10 (e.g., 12 months in patients without CEBPA mutations; P = ; P = ). AML patients with FLT3-ITD had significantly shorter median DFS (P = ) and OS (P = ) than patients without FLT3-ITD. High BAALC expression predicted for a shorter DFS (P = ) and OS (P = ) compared with AML with low BAALC expression; 53.7% of normal-karyotype AML had neither FLT3-ITD nor CEBPA Received 8/4/04; revised 11/12/04; accepted 11/16/04. Grant support: Swiss National Science Foundation grant (T. Pabst) and SAKK pilot project award The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: M. Bienz and M. Ludwig contributed equally to this work. Requests for reprints: Thomas Pabst, Institute of Medical Oncology, University Hospital of Berne, CH-3010 Berne, Switzerland. Phone: ; Fax: ; thomas.pabst@insel.ch. D2005 American Association for Cancer mutations. We found that high BAALC expression in normalkaryotype AML with neither FLT3-ITD nor CEBPA mutations (18 of 67) indicates adverse prognosis for both DFS and OS (P = ; e.g., P = ) compared with the group with low BAALC expression and absent FLT3-ITD and CEBPA mutations (18 of 67). Thus, BAALC expression represents a novel prognostic marker particularly for normal-karyotype AML patients with neither FLT3-ITD nor CEBPA mutations. Conclusions: Assessment of CEBPA mutations, FLT3- ITD, and BAALC expression permits to split normalkaryotype AML into clinically distinct subgroups. INTRODUCTION The karyotype of acute myeloid leukemia (AML) assessed at diagnosis is generally recognized as the single most valuable prognostic factor in AML (1, 2). However, using conventional cytogenetic techniques, karyotype abnormalities are detected in only half of all AML cases (1, 2), although the other half are commonly described as normal-karyotype AML. Patients with normal-karyotype AML usually have an intermediate risk with a 5-year overall survival of between 35% and 45% (1 3), but clinical outcome may vary greatly. In addition, the appropriate choice of consolidation in first remission (chemotherapy versus autologous transplantation versus allogenous transplantation) is unclear for these patients (1 3). Thus, additional markers with prognostic significance are needed to identify clinically relevant subgroups among AML patients with a normal karyotype. Some interesting candidate markers are now becoming available. The transcription factor CEBPA (for CCAAT/enhancer binding protein-a; for review, see ref. 4) is expressed in myelomonocytic cells and specifically up-regulated during granulocytic differentiation (5). cebpa knockout mice show a selective block in neutrophil differentiation at the stage of myeloblasts that is similar to the maturation arrest seen in human AML patients (6). Dominant-negative mutations of the CEBPA gene have been reported by us and others preferentially in AML patients with a normal karyotype and with myeloblastic AML subtypes (AML-M1 and M2; refs. 7 12). Interestingly, prognosis of these AML patients seems to be favorable (9 11). FLT3 (for fms-like tyrosine kinase 3) is a class III tyrosine kinase receptor (for review, see ref. 13). It is involved in signaling pathways regulating the proliferation of pluripotent stem cells and early progenitor cells. Internal FLT3 tandemly duplicated sequences (ITD) within the JM domain encoded by exons 14 and 15 are the most frequent single mutation described in adult AML with a reported incidence between 13% and 32% (14 22). Remarkably, FLT3-ITD AML exhibit a high relapse risk, decreased disease-free survival (DFS) and overall survival (OS; refs. 17, 18). Several groups have found in multivariate analysis that in AML FLT3-ITD is the most significant factor predicting an adverse outcome (17, 18).

2 Clinical Cancer Research 1417 BAALC (for brain and acute leukemia, cytoplasmic) is a recently identified gene on chromosome 8q22.3 with a protein sequence showing no homology to any other known proteins or functional domains (23). In hematopoietic cells, BAALC expression is restricted to progenitor cells (23). BAALC expression is found in AML and chronic myelogenous leukemias in blast crisis whereas no BAALC expression could be detected in patients with chronic-phase chronic myelogenous leukemia (23). In AML patients with normal cytogenetics, high BAALC expression seems to predict a poor prognosis (24). However, the expression of the BAALC gene and its potential use as a prognostic marker in normal-karyotype AML in the absence of CEBPA or FLT3-ITD mutations are unknown. In the present study, we assessed the presence of mutations in the CEBPA gene, of FLT3-ITD, and of the expression of the BAALC gene in untreated AML with normal cytogenetics. We show that this panel of markers adds important prognostic information for this largest subgroup in AML. MATERIALS AND METHODS Patient Samples. The diagnosis of AML was made using standard morphology and immunophenotype markers. Leukemic cells were purified at diagnosis from 67 consecutive patients with AML of all subtypes from a single university center. Details of the patients are given in Table 1. All patients were treated in previously published or ongoing protocols (SAKK 30/95 and SAKK 30/00; ref. 25). Performance status had to be WHO 0-2, and no severe organ dysfunction was allowed. All patients had a normal karyotype and were thus considered standard risk. Treatment for these standard-risk patients in cycle 1 consisted of cytarabine and idarubicin and in cycle 2 of cytarabine and amsacrin. Patients in complete remission after cycle 2 were randomly assigned to a third cycle of chemotherapy with etoposide and mitoxantrone or high-dose chemotherapy with busulfan and cyclophosphamide followed by autologous stem cell transplantation. Allogeneic stem cell transplantation was done in first complete remission if a suitable sibling donor was available and the patient was younger than 55 years. Mutational and Expression Analysis. Total cellular RNA was extracted from Ficoll density gradient centrifugation enriched mononuclear cells using the QIAmp RNA Blood Mini Kit (Qiagen, Chatsworth, CA). cdna was synthesized from 2 Ag total RNA applying the Superscript system and random hexamer primers (Invitrogen, San Diego, CA). For CEBPA mutational analysis, the entire coding region of the gene was amplified using three (A, B, and C) overlapping PCR primer pairs as previously described (7). Sequences of the primers used are listed in Table 2. PCR products were verified on agarose gel electrophoresis, and sequenced in both directions using BigDye Terminator-Mix Version 3.1 (ABI, Rotkreuz, Switzerland). Abnormal sequencing results were repeated twice in both directions including repetitions of PCR. Analysis of the internal tandem duplication of the FLT3 gene was done by amplification of the JM domain located in exons 14 and 15 and subsequent gel electrophoresis. BAALC mrna expression (Table 2) was normalized to the simultaneously analyzed PBGD gene. The relative BAALC expression was determined using the comparative cycle threshold (C T ) method. The cycle number difference was calculated as (DC t = C tbaalc C tpbgd ) of each replicate. The mean value from the duplicate was calculated as A (DC t ) =(ADC t ) / 2 and expressed as 2 A(DCt). The BAALC-positive leukemic Kasumi cell line and a negative control were included in each assay. The BAALC and PBGD C t values were measured in duplicate for each patient. In addition, we determined BAALC mrna levels in both leukocytes from peripheral blood and bone marrow samples from 12 healthy volunteers. Immunophenotyping and Cytogenetic Analysis. A panel of monoclonal antibodies against myeloid lineage associated antigens, including CD9, CD11b, CD13, CD14, CD15, CD33, glycophorin, and myeloperoxidase; lymphoid lineage associated antigens, including CD2, CD3, CD7, CD10, CD19, CD22, CD79; and lineage-nonspecific antigens, including HLA-DR, TdT, CD34, CD45, and CD56 was used to analyze the leukemic cells. The cutoff for a positive result of a particular marker was set at >20%. All cytogenetic analyses were done at a single reference institution, at the university hospital of Lausanne, Switzerland. Metaphase chromosomes were banded by conventional banding technique and karyotyped according to the International System for Human Cytogenetic Nomenclature. A karyotype was considered normal if at least 20 metaphases remained without evidence of a clonal abnormality. Statistical Analysis. The primary end point was DFS; the secondary end point was overall survival (OS). DFS was defined as the time from achievement of complete remission to first appearance of progression/relapse, or death from any cause. OS was defined as the time from diagnosis to death. Patients alive without progression/relapse by the time of analysis were censored at the time of their last follow-up. Time-to-event curves were constructed according to the Kaplan-Meier method and were compared with the log-rank v 2 test. Correlation coefficient was specified as Pearson correlation (r). RESULTS CEBPA Mutations Define a Subset of Normal Karyotype Acute Myeloid Leukemia with Favorable Prognosis. Heterozygous mutations of the CEBPA gene were found in 12 of 67 AML with a normal karyotype (17.9%). Eight of these 12 AML patients (66.7%) had two or more detectable CEBPA mutations, and a total of 25 CEBPA mutations were identified (Table 3). Ten of the 12 patients with CEBPA mutations had frameshift mutations with truncation of the protein (Fig. 1). In six patients, in-frame insertions at the COOH-terminal in the basic leucine zipper domain were seen. A total of 10 point mutations were detected in five patients. In two patients, the point mutations created novel stop codons. Of particular interest is the 212C > A point mutation in patient 7 (Table 3). This mutation eliminates the serine at amino acid 21. Phosphorylation of Ser 21 has recently been shown crucial for CEBPA function, which in this patient is likely to be abolished (26). Interestingly, patients with CEBPA mutations differed in many aspects from AML patients with a normal karyotype and a wild-type CEBPA gene status (Table 1). Their leukocyte

3 1418 Prognostic Subgroups in AML All (N = 67) 49 (18-71) CEBPA mutation (n = 12) 46 (22-58) Table 1 CEBPA wild type (n = 55) P 49 (18-71) Presenting characteristics FLT3-ITD (n = 19) FLT3 wild type (n = 48) P 48 (18-71) BAALC high (n = 44) BAALC low (n = 23) P (19-70) Median, age, y (range, y) (21-69) (18-71) Sex 33f / 34m 5f /7m 28f / 27m f /11m 25f /23m f /25m 14f /9m WBC, 10 9 /L, median (range) ( ) ( ) ( ) ( ) ( ) ( ) ( ) % Blasts in blood, median (range) (1-98%) (26-94%) (1-98%) (24-98%) (1-95%) (7-98%) (1-95%) LDH units/l, median* De novo AML (%) Secondary AML (%) MDS/therapy-related (6 / 5) (1 / 0) (5 / 5) (1 / 0) (5 / 5) (4 / 5) (2 / 0) Source PB 39 (58%) 7 (58%) 32 (58%) (68%) 26 (57%) (66%) 10 (43%) BM 28 (42%) 5 (42%) 23 (42%) 6 (32%) 22 (43%) 15 (34%) 13 (57%) Consolidation CR1 y Chemotherapy 36 (63%) 7 (58%) 29 (66%) 10 (67%) 26 (62%) 20 (54%) 16 (80%) Autologous Tx 10 (17%) 3 (25%) 7 (15%) 2 (13%) 8 (19%) 7 (19%) 3 (15%) Allogenous Tx 11 (20%) 2 (17%) 9 (19%) 3 (20%) 8 (19%) 10 (27%) 1 (5%) FAB classification M M M M M M M Gingiva hyperplasia Lymphadenopathy Hepatomegaly Splenomegaly Lung infiltrates Skin infiltrates NOTE. Presenting characteristics of the patients analysed. Seventeen of the 67 patients were older than 60 years at diagnosis. Source indicates whether blood or bone marrow at diagnosis was analysed. Abbreviations: Tx, transplantation; FAB, French American British classification. *LDH normal <480 units/l. yfifty-seven of 67 patients achieved a first complete remission (CR1) and thus underwent consolidation therapy. count (WBC) at diagnosis tended to be lower (16.1 versus 29.1 G/L). In addition, the median lactate dehydrogenase (LDH) value at diagnosis was not elevated in patients with CEBPA mutations compared with patients without CEBPA mutations (368 versus 1074 units/l; P = ). Remarkably, immunophenotyping revealed that in 50% of patients with CEBPA mutations (6 of 12), leukemic cells expressed the lymphoid marker CD7 whereas none of the patients without CEBPA mutations expressed this marker (Table 4). This has not been reported before to our knowledge. As previously shown by us and others, CEBPA mutations were limited to the myeloblastic subtypes of AML (M1 and M2) but were absent in the monocytic (M4 and 5), erythroblastic (M6), and megakaryoblastic (M7) subtypes (17 22). The clinical course was remarkably different in AML patients with or without CEBPA mutations. A complete remission after induction chemotherapy was achieved in all 12 AML patients with CEBPA mutations but only in 82% of AML without mutations. Figure 2C and D illustrates that patients without CEBPA mutations had a significantly shorter median DFS (10 months; P = ) and OS (12 months; P = ) than patients with CEBPA mutations (33.5 months; e.g., 45.5 months) as summarized in Table 5. We conclude that AML patients with a normal karyotype and CEBPA mutations have a remarkably favorable course of their disease. Internal Tandem Duplications of the FLT3 Gene Define a Subset of Normal Karyotype Acute Myeloid Leukemia with Particularly Adverse Prognosis. Tandemly duplicated sequences within the JM domain of the FLT3 gene were detected in 19 of 67 AML with a normal karyotype (28.4%). Patients with Table 2 Gene FLT3-F FLT3-R CEBPA-A-F CEBPA-A-R CEBPA-B-F CEBPA-B-R CEBPA-C-F CEBPA-C-R BAALC probe BAALC-F BAALC-R PBGD probe PBGD-F PBGD-R Sequences of primers and probes used in this study Nucleotide sequence 5V-AGCAATTTAGGTATGAAAGCCAG-3V 5V-CCTTCCCAAACTCTAAATTTTCTCT-3V 5V-TCGCCATGCCGGGAGAACTCTAAC-3V 5V-AGCTGCTTGGCTTCATCCTCCT-3V 5V-CCGCTGGTGATCAAGCAGGA-3V 5V-CCGGTACTCGTTGCTGTTCT-3V 5V-CAAGGCCAAGAAGTCGGTGGACA-3V 5V-CACGGTCTGGGCAAGCCTCGAGAT-3V 5V-CTCTTTTAGCCTCTGTGG TCTGAAGGCCAT-3V 5V-GCCCTCTGACCCAGAAACAG-3V 5V-CTTTTGCAGGCATTCTCTTAGCA-3V 5V-CTCATCTTTGGGCTGTTTTCTTCCGCCT-3V 5V-GGCAATGCGGCTGCAA-3V 5V-GGGTACCCACGCGAATCAC-3V

4 Clinical Cancer Research 1419 Table 3 C/EBPA mutations in normal karyotype AML patients Patient Age, y FAB Base pair change Amino acid change M GC>TT A199L 1V M1 1167G>A G339S 2 40 M insCG Y138fsX160 2V M insCTG L ins M insC E59fsX107 3V M insGTC V ins 4 24 M2 551G>A A134A 4V M insGCCGCCCC P199fsX M insGC A29fsX M2 395del F82fsX159 6V M insAAG K ins 7 47 M1 212C>A S21Q 7V M1 213del P22fsX159 7VV M insTCT S ins M1 1083C>T Q312X 9 33 M2 672C>G L175V 9V M2 676C>T A176V 9VV M GG>TT G177F 9VVV M2 683C>T L178L 9VVVV M2 688C>A P180H 9VVVVV M2 692C>G Y181X 10* 22.1 M insTCT S ins M insT A91fsX M insC E59fsX107 12V M insGTC V ins NOTE. afsxb indicates a frame-shift mutation of the amino acid at position a inducing a stop codon at position b. Abbreviation: FAB, French American British classification. *Patient 11 had homozygous insertion. FLT3-ITD differed in various aspects from other AML patients with a normal karyotype (Table 1). Leucocytes at diagnosis were higher (38.6 versus 17.6 G/L; P = ) in patients with FLT3- ITD. Also, the median LDH value at diagnosis was markedly increased in patients with FLT3-ITD as compared with AML patients without FLT3-ITD (1,292 versus 784 units/l; P = ). No significant differences in the imunophenotype between patients with and without FLT3-ITD were observed (Table 4). The course of AML in patients with FLT3-ITD was unfavorable. Although the rate of complete remissions achieved after induction chemotherapy was similar in patients with and without FLT3-ITD (84% versus 85%), the median DFS in patients with FLT3-ITD was significantly shorter (8.0 versus 12.5 months; P = ). Also, OS of patients with FLT3-ITD was decreased as compared with normal-karyotype AML without FLT3-ITD (10.1 versus 15.5 months; P = ). In summary, the presence of FLT3-ITD in normal-karyotype AML seems to confer an adverse clinical course. High BAALC Expression in Normal Karyotype Acute Myeloid Leukemia Is Associated with Unfavorable Prognosis. No differences were detected for BAALC mrna levels between peripheral blood leukocytes and bone marrow cells from 12 healthy volunteers. Figure 3A depicts BAALC levels for all volunteers with the values for peripheral blood on the x-axis and for bone marrow on the y-axis. The correlation coefficient (Pearson) was r = indicating a strong correlation between BAALC mrna levels in blood and bone marrow in a given volunteer. Furthermore, the range of BAALC expression among the 12 volunteers was remarkably small (range, 0.03 and 0.15; median, 0.09). We used the median value of 0.09 of these 12 volunteers as cutoff. Therefore, a value above 0.09 was considered high expression, whereas a value below 0.09 qualified for low expression. Fig. 1 Diagram of proteins encoded by wildtype and mutant CEBPA alleles. Locations of the amino acids corresponding to transactivation (black bars) domain 1 (79-97) and domain 2 ( ), and the basic zipper domain ( ; black bar). In-frame initiation codons at amino acids 1 and 120 encoding proteins of 42 and 30 kda. A, proteins encoded by six NH 2 - terminal mutants. The mutant peptide contains wild-type CEBPA sequence (white bars or black bars) followed by a shift of the reading frame (shaded bars) encoding a novel peptide before termination at a novel stop codon. B, four mutants were located at the COOH-terminal encoding novel stop codons (8 and 9VVVVV) or leading to a frame shift with subsequent truncation by a novel stop codon (2 and 4V). C, these six mutants were in-frame insertions within the basic zipper domain. D, point mutations with changes of the amino acids were seen in four patients.

5 1420 Prognostic Subgroups in AML Table 4 Immunophenotypes CD7 CD11b CD34 CD15 MPO All (N = 67) CEBPA mutation (n = 12) CEBPA wild type (n = 55) FLT3-ITD (n = 19) FLT3 wild type (n = 48) BAALC high (n = 44) BAALC high without FLT3/ CEBPA mutation (n = 23) BAALC low (n = 23) BAALC low without FLT3/ CEBPA mutation (n = 15) NOTE. Correlation of immunophenotypes and the CEBPA mutation status, the FLT3-ITD, and BAALC expression. Numbers indicate percentages of patients in the respective group expressing a given marker. The cutoff for a positive result of a particular marker was set at >20%. Significant differences were seen for CD7 expression for patients with and without CEBPA mutations (P = ), and for CD11b (P = 0.019), CD34 (P = 0.001), CD15 (P = 0.044), and myeloperoxidase (P = 0.031) depending on high or low expression of BAALC. Differences for these markers were also significant between high and low BAALC expression when patients with FLT3-ITD or CEBPA mutations were excluded. No differences were detected in cells with FLT3 wild type versus ITD. No significant differences were detected in any groups for the lymphoid markers CD2, CD3, CD10, CD19, CD22, CD79, and the myeloid markers CD9, CD13, CD14, CD33, CD45, CD56, and for HLA-DR and TdT. BAALC expression levels in 67 normal-karyotype AML ranged from to Twenty-three of the 67 patients (34.3%) fulfilled the criteria for low BAALC expression, whereas 44 patients (65.7%) were classified as high BAALC expression. Again, we determined whether the BAALC mrna levels in blood and bone marrow correlated for a given patient. Indeed, Fig. 3B indicates a strong correlation (r = ) between BAALC levels in blood and bone marrow in the 29 patients in whom both bone marrow and blood were available at diagnosis. We also evaluated whether the percentage of blasts in a given sample correlated with BAALC expression levels. However, no correlation was found between the percentage of blasts and BAALC expression (Pearson correlation, r = ). Interestingly, patients with high BAALC expression did not differ significantly from patients with low BAALC expression in terms of leukocyte count at diagnosis and the median LDH value at diagnosis (Table 1). However, we observed that monoblastic subtypes of AML predominantly had low BAALC expression (80%; 12 of 15 AML-M4 and M5) as previously reported (24). Low BAALC expression was observed in myeloblastic AML (M1 and M2) in only 25.6% (11 of 43 patients). In contrast, high BAALC expression was predominantly observed in myeloblastic subtypes of AML (32 of 43; 74.4%) and undifferentiated AML (M0; 5 of 5). All AML with M6 and M7 subtypes (four patients) also had high BAALC expression. Interestingly, significant differences were detected in the immunophenotype depending on different BAALC expression. Leukemic cells with low BAALC expression had significantly higher expression of the CD11b, CD15, and myeloperoxidase antigens. In addition, low BAALC expression correlated with low CD34 expression (Table 4). The rate of complete remission achieved after induction chemotherapy was not different in patients with high versus low BAALC expression (82% versus 91%; P = ). However, median DFS in patients with high BAALC expression was significantly shorter (8.5 versus 21 months; P = ). Also, overall survival of patients with high BAALC expression was decreased as compared with patients with low BAALC expression (10 versus 21 months; P = ). In summary, high BAALC expression in normal-karyotype AML seems to be associated with shortened DFS and OS. In Fig. 4, dot blot representations of BAALC expression levels are depicted for all patients together (left column; n = 67), for patients with FLT3-ITD only (second column; n = 19), for patients with CEBPA mutations only (middle column; n = 12), for patients having neither FLT3-ITD nor CEBPA mutations (fourth column; n = 36), and finally for the control group of 12 healthy volunteers. We found that patients with FLT3-ITD had a broad range of BAALC expression. Because high and low BAALC expressing patients with FLT3-ITD did not differ in their course (data not shown), we conclude that the presence of FLT3-ITD outweighs the significance of BAALC expression. Interestingly, All (N = 67) Table 5 CEBPA mutation (n = 12) Effect of CEBPA, FLT3, and BAALC status on clinical outcome CEBPA wild type (n = 55) P FLT3-ITD (n = 19) FLT3 wild type (n = 48) P BAALC high (n = 44) BAALC low (n = 23) P CR1 achieved, % Death in induction, % Death in CR1 (n) Relapse, % OS median, mos (range, mos) (0-166) (20-166) (0-113) (0-103) (0-166) (0-103) (0-166) DFS median, mos (range mos) (0-136) (9-136) (0-113) (0-103) (0-136) (0-136) (0-113) DFS at three years, (n) OS at three years, (n) Patients disease-free in follow-up*, n Median, mos Range, mos NOTE. No patient died during consolidation therapy randomized to chemotherapy (n = 36) or to autologous transplantation (n = 10), and one patient died during allogeneic transplantation (1 of 11). DFS and OS are given as a median and also at the time point 3 years after diagnosis. *Duration of follow-up is indicated for patients who are still disease free with median value and range.

6 Clinical Cancer Research 1421 patients with CEBPA mutations were predominantly seen in the high BAALC expression group. Only three out of 12 patients with CEBPA mutations relapsed. Remarkably, these three patients showed the highest BAALC expression among the group of 12 normal-karyotype AML patients with CEBPA mutations. We also analyzed within the high BAALC expression group whether DFS or OS in patients with very high expression (top 50%) was shorter than in patients with only high expression (lower 50%). However, patients with very high BAALC expression did not differ in their DFS and OS from patients with high BAALC expression (P = and P = 0.757, respectively.). Similarly, we studied within the low BAALC expression group whether clinical outcome in patients with very low expression was more favorable than in patients with only low expression. Again, we observed that patients with very low BAALC expression did not differ in their DFS and OS from patients with low BAALC expression (P = and P = 0.746, respectively). These results support the usefulness of our cutoff indicating that this cutoff indeed seems to separate groups of prognostic favorable and unfavorable patients. Low BAALC Expression in Normal Karyotype Acute Myeloid Leukemia with neither CEBPA nor FLT3 -ITD Mutations Is Associated with Favorable Prognosis. We hypothesized that determining BAALC expression might be Fig. 2 DFS (A, C, and E ) and OS (B, D, and F) in normalkaryotype AML patients according to the FLT3-ITD status (A-B), the CEBPA mutation status (C-D ), and the BAALC expression (E-F).

7 1422 Prognostic Subgroups in AML Fig. 3 Correlation of BAALC mrna levels in peripheral blood (PB) leukocytes (x-axis) and bone marrow (BM) cells (y-axis) from 12 healthy volunteers (A). Pearson correlation coefficient r = indicates strong correlation between BAALC mrna levels in blood and bone marrow. B, BAALC mrna levels in peripheral blood (x-axis) and bone marrow cells (y-axis) from 29 normalkaryotype AML patients at diagnosis with r = again suggesting a strong correlation. particularly useful in the subset of AML patients with neither FLT3-ITD nor CEBPA mutations (36 of 67). The median BAALC expression of patients in this subgroup was about 10- fold higher than the value of our control group. Eighteen of these 36 patients (50%) had low BAALC expression, and 18 had high BAALC expression. Most interestingly, the clinical course of these two subgroups differed dramatically both for DFS (18.4 and 7.4 months, respectively; P = ) and for OS (22.8 and 9.1 months, respectively; P = ) as depicted in Fig. 5A and B. We thus conclude that BAALC expression adds significant prognostic information particularly in those AML patients with a normal karyotype where until now other markers such as FLT3-ITD or CEBPA mutations are lacking. Ultimately, we did a multivariable analysis to investigate whether CEBPA mutations, FLT3-ITD and BAALC expression represent independent prognostic markers in normal-karyotype AML. The results of this analysis are summarized in Table 6 indicating that CEBPA mutations, FLT3-ITD, and BAALC expression seem to be strong independent predictors of outcome in normal-karyotype AML. DISCUSSION The prognostic effect of various chromosomal aberrations in AML is well established with implications for therapy. In our well-defined cohort of 67 consecutive normal-karyotype AML patients, we have now identified three independent molecular prognostic factors that define distinct subgroups. The presence of mutations in the CEBPA gene indicated a favorable course of the disease, whereas FLT3-ITD confers a bad prognosis. In addition, high expression of BAALC mrna is associated with a significantly worse prognosis. We detected CEBPA mutations in one sixth of normalkaryotype AML patients. Others have reported slightly lower percentages with between 4.3% and 11% (7 10, 12). However, most of these studies have not focussed on normal-karyotype AML. In accordance with others, we confirmed that CEBPA mutations preferably occur in the FAB classes M1 and M2 (7 12). The clinical effect of CEBPA mutations seems to be distinctly favorable. Our AML patients with CEBPA mutations had a median DFS of 33.5 months and an OS of 45.5 months. In parallel with accepted practice in other types of good-prognosis AML such as acute promyelocytic leukemia or AML with abnormalities

8 Clinical Cancer Research 1423 Fig. 4 Dot plots representing individual levels of BAALC expression. Median BAALC values (line). All, BAALC values of all 67 normalkaryotype AML patients; FLT3, group of patients with FLT3-ITD (n = 19); CEBPA, group of patients with CEBPA mutations (n = 12), w/o mut, group of patients with neither FLT3-ITD nor CEBPA mutations (n = 36); normal, BAALC values of 12 healthy volunteers. of chromosome 16, patients with CEBPA mutations and a normal karyotype may enjoy long-lasting first remission without allogeneic stem cell transplantation. Several groups reported a significantly increased frequency of FLT3-ITD in normal-karyotype AML compared with other AML subgroups (15, 17, 20). Our data are in accordance with this literature. The most significant effect of an FLT3-ITD on clinical outcome is its association with increased relapse risk, decreased DFS and OS (13). Several groups reported that an ITD is the most significant factor predicting an adverse outcome in multivariate analysis (17, 18). In our series of normal-karyotype AML, patients with FLT3-ITD had a significantly reduced DFS and OS. Thus, normal-karyotype AML patients with FLT3-ITD seem to represent the unfavorable end of the prognostic spectrum, with patients with CEBPA mutations representing the favorable end. In our series, 53.7% of normal-karyotype AML patients had neither CEBPA mutations nor FLT3-ITD. We now propose to assess BAALC expression to obtain further prognostic information particularly in those patients. In a recent report investigating 86 de novo AML with a normal karyotype, high BAALC expression was found associated with significantly decreased OS and DFS (24). In the study cited above, patients were dichotomized at BAALC s median expression into low and high Fig. 5 DFS (top) and OS (bottom) of normal-karyotype AML patients with neither FLT3-ITD nor CEBPA mutations (n = 36) according to their BAALC expression (high, n = 18; low, n = 18). expressers. This approach might produce varying levels of median expression depending on the collection of patients studied. We used a slightly different approach. Bone marrow and peripheral blood samples from 12 healthy volunteers were analyzed for BAALC expression. We found a very small range of expression within this normal control group with no differences Table 6 Multivariable analysis for overall survival and disease-free survival OS Hazard ratio (95% confidence interval) P DFS Hazard ratio (95% CI) P CEBPA mutation ( ) ( ) FLT3 wild type ( ) ( ) high BAALC ( ) ( ) Age ( ) ( ) WBC ( ) ( ) LDH ( ) ( ) NOTE. Hazard ratios and P values are given for CEBPA mutations versus wild type, FLT3 wild type versus FLT3-ITD, and high versus low BAALC expression, as well as for age, WBC, and LDH.

9 1424 Prognostic Subgroups in AML between peripheral blood and bone marrow samples. The median expression value of these controls was used as cutoff. Patients were then classified into two subgroups with either BAALC expression above or below this normal BAALC expression level. Most interestingly, normal-karyotype AML patients with low BAALC expression had a significantly better clinical outcome than high expressers both for DFS and OS. In summary, we identified CEBPA mutations, FLT3-ITD, and differing levels of BAALC expression as having independent prognostic significance in normal-karyotype AML. We propose that molecular assessment of these three factors at diagnosis offers valuable additional prognostic information and may thereby markedly affect therapeutic decisions. REFERENCES 1. Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 Trial. 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10 Risk Assessment in Patients with Acute Myeloid Leukemia and a Normal Karyotype Marianne Bienz, Madleina Ludwig, Beatrice U. Mueller, et al. Clin Cancer Res 2005;11: Updated version Access the most recent version of this article at: Cited articles Citing articles This article cites 13 articles, 2 of which you can access for free at: This article has been cited by 37 HighWire-hosted articles. Access the articles at: alerts Sign up to receive free -alerts related to this article or journal. Reprints and Subscriptions Permissions To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, use this link Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.