Document YrQgb8o5rqX4Lg8b2k5Q58KrN

Comparison of Cytogenetic and Molecular Cytogenetic Detection of Chromosome Abnormalities in 240 Consecutive Adult Patients With Acute Myeloid Leukemia By Stefan Frohling, Silvia Skelin, Claudia Liebisch, Claudia Scholl, Richard F. Schlenk, Hartmut Dohner, and Konstanze Dohner for the Acute Myeloid Leukemia Study Group Ulm Purpose: To prospectively compare cytogenetic and molecular cytogenetic analysis for the detection of the most relevant chromosome abnormalities in a large series of patients with acute myeloid leukemia (AML). Patients and Methods: Two hundred forty consecutive adult patients with AML entered onto the multicenter treatment trial AML HD93 were studied. Chromosome banding and fluorescence in situ hybridization (FISH) applying a comprehensive set of genomic DNA probes were performed in a single reference laboratory. Results: Two cases of inv(16), three cases of t(11q23), and three cases of t(8;21)var were only detected by molecular cytogenetics. By FISH, aberrations were identified in three cases with normal karyotypes: inv(16), Y (in a patient with low metaphase yield on chromosome banding) and a 12p microdeletion. Additional aneuploidies, in particular 8q and 11q, were diagnosed by FISH; however, virtually all these aber- rations occurred in patients with complex karyotypes or as an additional abnormality in leukemias with an AML-specific translocation. Finally, aberrations were detected by FISH in eight of 14 patients with no assessable metaphases. Conclusion: In most cases of AML, conventional cytogenetic study reliably detects chromosomal abnormalities, and this method should not be replaced by FISH. FISH should be used as a complementary method for the detection of more subtle abnormalities, such as inv(16) and t(11q23), in all patients with newly diagnosed AML and for suspected t(8;21)var. Furthermore, molecular cytogenetics using this comprehensive set of DNA probes provides a valuable diagnostic tool for patients with poor chromosome morphology, low or no yields of metaphase cells, or both. J Clin Oncol 20:2480-2485. 2002 by American Society of Clinical Oncology. I N ACUTE MYELOID leukemia (AML), clonal chromosome aberrations constitute markers of diagnostic value, and the molecular characterization of numerous abnormalities has greatly improved the understanding of the biology of distinct subtypes of the disease.1-4 Moreover, cytogenetic data are particularly useful for determining prognosis or predicting response to certain therapeutic approaches.5-7 Thus, because modern risk-adapted therapy of patients with AML is based on cytogenetic characteristics, the rapid and reliable identification of chromosome aberrations is of utmost importance for the clinical management of patients with this malignancy. Conventional chromosome banding study allows a comprehensive analysis of the karyotype and is still a fundamental component of modern tumor cytogenetics. Nevertheless, despite continuous improvements in cytogenetic From the Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany. Submitted August 23, 2001; accepted February 20, 2002. Supported in part by grant no. 98.025.1 from the Wilhelm Sander-Stiftung. Address reprint requests to Konstanze Dohner, MD, Department of Internal Medicine III, University Hospital of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany; email: konstanze.doehner@medizin.uni-ulm.de. 2002 by American Society of Clinical Oncology. 0732-183X/02/2010-2480/$20.00 methodology, in almost all studies of patients with AML, substantial numbers of patients without adequately banded metaphase cells have been reported.1,2 With the increasing availability of a large variety of specific DNA probes due to the developments in the Human Genome Project (http://www.ncbi.nlm.nih.gov), studies that use fluorescence in situ hybridization (FISH) are no longer limited to the detection of numerical aberrations or the identification of certain structural abnormalities in a small number of patients.8 In 1996, our group reported on the use of FISH for a comprehensive analysis of the most relevant AML-associated chromosome aberrations in a pivotal series of 105 patients with adult AML.9 FISH proved to be a rapid and sensitive technique that complemented conventional chromosome banding studies. We present the results from a comparative study of cytogenetic and molecular cytogenetic analysis in a prospective series of 240 patients with adult AML entered onto the German multicenter treatment trial AML HD93. PATIENTS AND METHODS Patients Between December 1993 and January 1998, 240 consecutive patients (aged 16 to 60 years) with AML (de novo and secondary after a primary malignancy) diagnosed according to French-American-British Cooperative Group criteria10 were studied by chromosome banding analysis and FISH in our laboratory, which is the central reference 2480 Journal of Clinical Oncology, Vol 20, No 10 (May 15), 2002: pp 2480-2485 DOI: 10.1200/JCO.2002.08.155 Downloaded from jco.ascopubs.org on April 29, 2010 . For personal use only. No other uses without permission. Copyright 2002 by the American Society of Clinical Oncology. All rights reserved. DETECTION OF GENOMIC ABERRATIONS IN AML Table 1. Designation, Chromosomal Localization, and Gene/Locus of the DNA Clones Selected for the Detection of AML-Associated Aberrations by FISH Aberration DNA Clone Localization Gene/Locus 5/5q 7/7q 8/8q t(8;21) 9/9q t(11q23) t(9;11) t(12p13)/12p 13q/13q t(15;17) inv(16) 17/17p 20/20q 21/21q 22/22q /Xq /Yq 773_D_3 yPR411 HSC7E506 HSC7E124 935_A_12 464_H_8/72_H_9 P1 164 phage 253_F_8 933_G_6 13HH4 785_C_6/856_B_9 48/55 cosmid pool 964_C_10 RB1 phage pool 804_D_9 356_C_12 RARAcos121/124 Cosmid pool 854_E_2 LA2-2/LA4-1 cosmid pool TP53 cosmid pool 808_C_05 464_H_8/72_H_9 2D3 D107F9 361_D_9 A24CH4 IE018 yOX-57 5q31 5q33 7q22 7q35 8q24 21q22 8q22 9q13-q21 9q22.3 11q23 11q23 9p22 12p13 13q14 13q14 15q22 17q21 16p13 16q22 17p13 20q13 21q22 21q, proximal half 22q11 22q11 Xq28 Xq28 Yq11 IL9, D5S89 CSF1R RELN, D7S240 D7S688 D8S508 AML 1 ETO D9S15 D9S280 MLL MLL, Thy 1 AF9 TEL RB1 RB1 PML RARA MYH11 CBF TP53 D20S99 AML 1 BCR BCR DXS304 DXS304 DXYS1 laboratory for cytogenetic and molecular diagnostics within the AML Study Group Ulm. All patients were entered into the multicenter treatment trial AML HD93, in which postremission therapy was stratified according to the karyotype11: low risk [t(8;21), inv(16)/t(16; 16), t(15;17)], intermediate risk (normal karyotype), and high risk (all other chromosome aberrations). Conventional chromosome banding studies were performed using standard techniques and chromosomal abnormalities were described according to the International System for Cytogenetic Nomenclature.12 The cytogenetic risk classification defined all patients with t(8;21), inv(16)/t(16;16), or t(15;17) as favorable, regardless of additional abnormalities. Complex karyotypes were defined as the presence of three or more clonal aberrations. The study was approved by the institutional review boards of the participating institutions. Informed consent was obtained from all patients according to the guidelines set forth by institutions participating in the study. Molecular Cytogenetic Analysis The DNA clones selected for the detection of the most relevant AML-associated chromosome aberrations by FISH (Table 1), the criteria required for the identification of AML-specific gene fusions, and the cutoff levels for the diagnosis of aneuploidies or deletions were 2481 described previously.9 In the course of this study, the DNA probe set was subjected to several modifications: because of the heterogeneity of the breakpoints at 9q reported in the literature,1,2 yeast artificial chromosome (YAC) clone 933_G_6 was included to identify deletions telomeric to 9q21. The RB1 phage pool used to detect deletions at 13q14 was replaced by YAC clone 804_D_9 because this probe yielded a greater fluorescence signal intensity. YAC clone 2D3 mapping to the proximal half of 21q13 was added to the probe set to distinguish between translocations involving the AML1 gene at 21q22, as detected by the 464_H_8/72_H_9 YAC pool and trisomy 21q. YAC clone 361_D_9 was used to detect disruption of the BCR gene at 22q11 because a greater proportion of Philadelphia chromosomepositive patients exhibited splitting of one fluorescence signal with this probe than with D107F9. In addition to the DNA clones listed in Table 1, centromere-specific probes were used to distinguish between monosomies or trisomies, and partial aneuploidies. Cosmid and P1 phage DNA was prepared using the Plasmid Midi Kit (Qiagen, Hilden, Germany). Human sequences from YAC clones were generated by an inter-Alu polymerase chain reaction (PCR) protocol.14 The sequence-independent amplification technique15 was used to amplify DNA from YAC clone 854_E_2 and from RB1 phage clones. PCR products were labeled by nick translation with biotin-16-dUTP or digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany). Dual-color FISH was performed as previously described.9 The hybridization mixture contained approximately 250 ng labeled cosmid, P1 phage or YAC DNA, 10 g Cot-1 DNA fraction (BRL/Life Technologies, Gaithersburg, MD) and 10 g herring sperm DNA (Roche Diagnostics). FISH signals were visualized by a Zeiss Axioskop epifluorescence microscope (Oberkochen, Germany), and images were captured by a cooled charged coupled device camera (Xillix Technologies, Richmond, Canada). RESULTS Two hundred forty patients were studied by cytogenetics and FISH. One hundred five of these patients have been reported previously.9 Results from the two methods were compared with regard to the detection of the most relevant AML-specific gene fusions and the most frequent aneuploidies or deletions. Because karyotype was used for selecting postremission therapy within the AML HD93 treatment trial, results of all patients were compared, irrespective of success of the assays. The distribution of individual aberrations is listed in Table 2. The combined results of cytogenetic and molecular cytogenetic analysis are provided in Table 3. Assessable metaphases were obtained in 226 (94%) of 240 patients. Of the 226 assessable patients, 124 (55%) exhibited clonal chromosome aberrations; 102 patients (45%) had a normal karyotype. In contrast to chromosome banding analysis, assessable interphase preparations were obtained in all 240 patients. Clonal aberrations were detected in 125 patients (52%). Translocations and Inversion All patients with a t(8;21)(q22;q22) on banding analysis and four additional cases of an AML1/ETO fusion were detected by FISH; three of these four patients had a variant Downloaded from jco.ascopubs.org on April 29, 2010 . For personal use only. No other uses without permission. Copyright 2002 by the American Society of Clinical Oncology. All rights reserved. 2482 Table 2. Distribution of AML-Specific Aberrations in 240 Consecutive Adult Patients: Comparison of Cytogenetic and Molecular Cytogenetic Analysis Aberration Chromosome Banding No. of Aberrations FISH Chromosome Banding or FISH Translocations/inversion t(8;21)/t(8;21)var 13 17 17 t(9;11) 11 11 11 t(11q23) other 699 t(12p13) 222 t(15;17) 18 18 19 inv(16)/t(16;16) 22 25 25 Aneuploidies/deletions 5/5q 14 14 15 7/7q 18 17 19 8/8q 16 25 25 9/9q 878 11/11q 6 12 12 12/12p 446 13/13q 445 17/17p 588 20/20q 434 21/21q 8 8 10 22/22q 588 X/Xq 688 Y/Yq 445 t(8;21), designated add(2)(p13), add(21)(q22), t(8;9; 21)(q22;q36;q22), and t(4;21)(p12;p11) on banding analysis; in one case, there were no assessable metaphases. Inv(16)(p13q22) or t(16;16)(p13;q22) was identified in 22 patients by cytogenetics and in 25 patients by molecular cytogenetics. The three additional patients identified by FISH included one patient with a normal karyotype, a second patient with numerical aberrations of chromosomes Y, 13, and 22 but without abnormality of chromosome 16, and a third patient without assessable metaphases. Of the 18 patients with t(15;17) found on banding analysis, one was not detected by molecular cytogenetics; and one patient without assessable metaphases demonstrated a t(15;17) by FISH. Table 3. Incidence of Clonal Chromosome Aberrations by Cytogenetics and Molecular Cytogenetics in 240 Consecutive Adult Patients With AML* Aberration Chromosome Banding No. % FISH No. % Chromosome Banding or FISH No. % Not assessable 14 6 None None No clonal aberrations 102 42 115 48 105 44 Clonal aberrations 124 52 125 52 135 56 *Percentages are based on all 240 cases. FRO HLING ET AL Rearrangements involving band 11q23 were identified in three additional patients by molecular cytogenetics: one patient had a ?del(11)(q23) on banding analysis, whereas interphase and metaphase FISH demonstrated a t(11; 16)(q23;p13). The second patient had an add(10)(q26) on banding analysis; molecular cytogenetic analysis using DNA probes recognizing band 11q23 (13HH4, 785_C_6/ 856_B_9 pool) displayed three fluorescence signals in 49% of the cells, and metaphase FISH revealed a t(10;11). In the third patient without abnormality of chromosome 11 on banding analysis, FISH that used the 785_C_6/856_B_9 pool and a chromosome 6 specific painting probe demonstrated that 11q23 material was inserted into band 6q28. Finally, both patients with balanced translocations involving chromosome band 12p13 were identified by cytogenetic and molecular cytogenetic analysis. Aneuploidies/Deletions FISH identified all cases of 5 and one additional case of 5q in a patient without assessable metaphases. One patient with a 5q within a complex karyotype was not detected by molecular cytogenetics using YAC clones 773_D_3 and yPR411. Metaphase FISH demonstrated that a translocation of chromosome 5 material to chromosome 19 had occurred: the D5S89 locus (identified by clone 773_D_3) was retained on the der(5), whereas the CSFR1 gene (identified by clone yPR411) was translocated to the der(19). Additional experiments that used YAC clone 802_D_5 mapping distally to 773_D_3 to band 5q31 revealed that this translocation was associated with a 5q that was missed by the DNA probes selected for this study. Of the six patients with 7 found on banding analysis, one with a subclone of 7 within a complex karyotype was not identified by FISH. One patient with a del(7)(q22q32) present in two of 16 metaphase cells analyzed also had no aberrations on molecular cytogenetic analysis. In one patient without assessable metaphases, a 7q was detected by FISH. All patients with 8/8q found on banding analysis and nine additional patients were identified by FISH: in two patients, a subclone of 8q was masked in a complex karyotype, one patient exhibited an i(10q) and a t(11q23), one patient had an add(7)(q3?1) and a t(15;17), and in five of these nine patients, no metaphase cells were obtained. In two of the five patients without assessable metaphases, 8/8q was the sole cytogenetic abnormality. Molecular cytogenetic analysis detected all cases of 11/11q found on banding analysis and six additional cases: in three patients without abnormalities of chromosome 11 on banding analysis and three patients without assessable metaphase spreads, FISH experiments demonstrated a 11q. In five of the six additional cases, the Downloaded from jco.ascopubs.org on April 29, 2010 . For personal use only. No other uses without permission. Copyright 2002 by the American Society of Clinical Oncology. All rights reserved. DETECTION OF GENOMIC ABERRATIONS IN AML 11/11q was part of a complex karyotype; one patient had a t(15;17) as the primary cytogenetic abnormality. Similarly, two additional cases of 12p were detected by FISH: one patient had a normal karyotype, and one patient had a t(11q23) as the sole cytogenetic abnormality on banding analysis. Of the three patients who had an add(12p) within a complex karyotype, only one was identified by FISH. Deletion of the short arm of chromosome 17 was detected in five patients by banding analysis and in eight patients by FISH. The three additional patients included two with complex karyotypes and one without assessable metaphases. The latter patient exhibited a complex karyotype on molecular cytogenetic analysis. Of the eight patients with trisomy 21 detected by chromosome banding analysis, two with subclones of 21 were missed by FISH: one patient had trisomy 8 and a t(11q23) as the leading abnormalities; one patient exhibited an inv(16). In contrast, two additional patients with 21q were identified by FISH: one patient had a complex karyotype without abnormality of chromosome 21, and in one patient, there were no assessable metaphases. Patients With Normal Karyotypes One hundred two patients had a normal karyotype on banding analysis. In three of these patients, single chromosome aberrations were detected by FISH: inv(16), Y, and 12p. In retrospective analysis of the first patient, the inv(16) was likely present but was missed as a result of poor chromosome morphology. In the patient with loss of the Y chromosome, only five metaphase spreads could be obtained; by FISH, 31% of the cells exhibited Y. In the patient with 12p, metaphase FISH demonstrated a normal size of the short arm of chromosome 12 but loss of the sequences detected by probe 964_C_10, indicating a microdeletion that was not detectable by chromosome banding analysis. Patients Without Assessable Metaphase Cells In 14 patients, no assessable metaphase preparations were obtained. In eight of these patients, chromosome aberrations were detected by FISH: t(8;21); 8, 9q, inv(16); t(15;17); 8 (two patients); 7q, 11q, 13q, Y; 7q, 8q, 11q, 17p, 21q; and 5q, 8, 11q. DISCUSSION The results from this study demonstrate the applicability of FISH for the detection of the most relevant AMLassociated chromosome aberrations in a large prospective series of patients with adult AML entered onto a multicenter treatment trial. The study was initiated to improve the 2483 accuracy of cytogenetic diagnostics within the AML HD93 trial in which pretreatment cytogenetics was used for selecting risk-adapted postremission therapy. All experiments were performed in a central reference laboratory, and FISH proved to be a rapid (results were available within 5 working days) and reliable (experiments were successful in all patients) technique. FISH was particularly sensitive for detecting the AML-specific translocations and inversion and some aneuploidies and deletions. Furthermore, FISH identified chromosome aberrations in three patients with normal karyotypes and eight of 14 patients in whom chromosome banding analysis was unsuccessful. Results from several trials suggest that two of the chromosomal rearrangements involving components of the core-binding factor transcription factor complex, inv(16) and t(8;21), confer a favorable prognosis, and it has been demonstrated that the outcome of patients with these aberrations can be significantly improved by the use of highdose cytarabine in postremission therapy.1,2,5,6,16 In our study, three additional cases of inv(16)(p13q22) were identified by FISH. These included one patient without assessable metaphases and two patients that had been missed on banding analysis, presumably because of insufficient chromosome morphology. In consequence of the low number of G bands within the inverted region of chromosome 16, high quality of metaphases is required for the definitive cytogenetic diagnosis of an inv(16). Therefore, the detection of this abnormality may be more difficult in the setting of a multicenter trial where the specimens are transported to a reference laboratory. Four additional cases of an AML1/ETO fusion were detected by FISH: in one patient, no assessable metaphases were obtained, and in three patients, the chimeric gene resulted from a variant t(8;21). Thus, because the classical t(8;21)(q22;q22) is relatively easy to detect at the cytogenetic level, FISH that uses probes for the identification of the AML1/ETO hybrid gene should be performed only in cases exhibiting breakpoints in bands 8q22 and/or 21q22, which may result from a variant t(8;21). Our findings are strikingly consistent with the results from a study of the Cancer and Leukemia Group B comparing cytogenetics and reverse transcriptasePCR for the detection of t(8;21)(q22; q22) and inv(16)(p13q22)/t(16;16)(p13;q22) in 284 patients with adult AML17: in this study, cytogenetic results were considered to be false negative in three AML1/ETO-positive patients with variants of t(8;21) and in three CBF/MYH11positive patients with, respectively, an isolated 22; del(16)(q22),22; and a normal karyotype. Reciprocal translocations involving the retinoic acid receptor alpha (RARA) gene include the t(15;17)(q22;q21), resulting in the juxtaposition of the PML gene and the RARA Downloaded from jco.ascopubs.org on April 29, 2010 . For personal use only. No other uses without permission. Copyright 2002 by the American Society of Clinical Oncology. All rights reserved. 2484 FRO HLING ET AL gene and the less frequent t(11;17)(q23;q21), t(11;17)(q13; q21), and t(5;17)(q35;q21), whereby RARA is fused to the PLZF, NuMA, and NPM genes, respectively.18 The majority of AML cases with morphologic and clinical features of acute promyelocytic leukemia (FAB M3) have a t(15;17). Detection of a t(15;17) is crucial in determining which individuals will derive therapeutic benefit from all-trans retinoic acid induction or maintenance therapy.19 In our hands, FISH offered no additional benefit for the detection of a t(15;17) in patients with successful banding analysis. Compared with inv(16), t(8;21), and t(15;17), the outcome of patients with chromosomal rearrangements involving the MLL gene at chromosome band 11q23 is less well defined. Complete remission rates of adult patients with abnormalities of 11q23 were reported to vary between 25% and 83%, possibly reflecting the marked heterogeneity of 11q23 aberrations, and overall survival of the majority of patients is poor.1,2,4 In the present study, all cases of t(9;11) were detected by FISH. This aberration is regarded to confer a more favorable prognosis than other 11q23 translocations, especially in patients who receive intensive postremission therapy with high-dose cytarabine or allogeneic bone marrow transplantation.6,20 In addition, three cases of other rearrangements involving band 11q23 were only detected by FISH using a YAC clone containing the entire MLL gene and a contig of YAC clones recognizing more than 1.0 Mb of DNA sequences distal to MLL. By FISH, more cases of genomic imbalances, in particular 8q and 11q, were detected. However, almost all aneuploidies or deletions that were missed on conventional cytogenetics were detected in patients with complex karyotypes or as additional abnormalities in patients with at least one leukemia-specific primary chromosome change--for example, inv(16), t(8;21), t(15;17), or t(11q23). In addition, some aneuploidies or deletions were identified in patients with normal cytogenetics or in patients without assessable metaphases. Considering that the majority of studies associate complex cytogenetic abnormalities with an adverse prognosis1,2 and that additional aberrations did not have a deleterious effect on outcome in a large series of patients with low-risk abnormalities entered into the 10th United Kingdom Medical Research Council trial,6 our data imply that routine FISH that uses DNA probes for the detection of chromosomal aneuploidies or deletions does not add prognostic information to that gained from pretreatment cytogenetics in patients in whom banding analysis is successful. On the contrary, patients without assessable metaphases should be screened for 5q, 7q, 8q, 12p, 17p, and 20q by molecular cytogenetics, particularly with regard to the dismal prognosis associated with these aberrations.1,2 FISH identified clonal aberrations in three of 102 patients with normal cytogenetics: inv(16); Y; and 12p. In the patient with inv(16), chromosome morphology was poor; in the Y patient, only five assessable metaphases could be obtained. Accordingly, the 12p was the only submicroscopic aberration detected by FISH in 102 patients with normal cytogenetics. These data indicate that molecular cytogenetics using the comprehensive probe set of our study does not add relevant information to that gained from conventional chromosome banding in patients with normal cytogenetics. Considering the heterogeneity of these leukemias at the molecular level,21-23 other techniques, such as Southern blot analysis, PCR, and DNA sequencing should be used to detect genetic abnormalities not resolvable by conventional banding analysis. Our study demonstrates that in the majority of cases, chromosome banding analysis, provided that sufficient metaphase cells are assessable, reliably yields the leukemia karyotype. We propose to use FISH as a complementary method for the identification of inv(16) and t(11q23) in all patients with newly diagnosed AML because these abnormalities may be difficult to detect and in patients suspected of having a variant t(8;21). Furthermore, molecular cytogenetics using the probe set presented in this study provides a valuable tool for patients with poor chromosome morphology and those with a low or no yield of assessable metaphase cells. Our study does not support routine screening by FISH of patients with normal karyotypes. ACKNOWLEDGMENT We thank the members of the AML Study Group Ulm for providing leukemia specimens. We also thank Brigitte Schreiter for technical assistance. REFERENCES 1. Mrozek K, Heinonen K, Bloomfield CD: Clinical importance of cytogenetics in acute myeloid leukaemia. Baillieres Best Pract Res Clin Haematol 14:19-47, 2001 2. Mrozek K, Heinonen K, de la Chapelle A, et al: Clinical significance of cytogenetics in acute myeloid leukemia. Semin Oncol 24:17-31, 1997 3. Rowley JD: The role of chromosome translocations in leukemogenesis. Semin Hematol 36:59-72, 1999 (suppl 7) 4. Willman CL: Molecular evaluation of acute myeloid leukemia. Semin Hematol 36:390-400, 1999 5. Bloomfield CD, Lawrence D, Byrd JC, et al: Frequency of prolonged remission duration after high-dose cytarabine intensification Downloaded from jco.ascopubs.org on April 29, 2010 . For personal use only. No other uses without permission. Copyright 2002 by the American Society of Clinical Oncology. All rights reserved. DETECTION OF GENOMIC ABERRATIONS IN AML 2485 in acute myeloid leukemia varies by cytogenetic subgroup. Cancer Res 58:4173-4179, 1998 6. Grimwade D, Walker H, Oliver E, et al: The importance of diagnostic cytogenetics on outcome in AML: Analysis of 1612 patients entered into the MRC AML10 trial. Blood 92:2322-2333, 1998 7. 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Mrozek K, Prior TW, Edwards C, et al: Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv (16) in a prospective series of adults with de novo acute myeloid leukemia: A Cancer and Leukemia Group B study. J Clin Oncol 19:2482-2492, 2001 18. Grimwade D, Biondi A, Mozziconacci MJ, et al: Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): Results of the European Working Party. Blood 96:1297-1308, 2000 19. Fenaux P, Chomienne C, Degos L: All-trans retinoic acid and chemotherapy in the treatment of acute promyelocytic leukemia. Semin Hematol 38:13-25, 2001 20. Mrozek K, Heinonen K, Lawrence D, et al: Adult patients with de novo acute myeloid leukemia and t(9;11)(p22;q23) have a superior outcome to patients with other translocations involving band 11q23: A Cancer and Leukemia Group B study. Blood 90:4532-4538, 1997 21. Caligiuri MA, Strout MP, Lawrence D, et al: Rearrangement of ALL1 (MLL) in acute myeloid leukemia with normal cytogenetics. Cancer Res 58:55-59, 1998 22. Kiyoi H, Naoe T, Nakano Y, et al: Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood 93:3074-3080, 1999 23. Pabst T, Mueller BU, Zhang P, et al: Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding proteinalpha (C/EBPalpha) in acute myeloid leukemia. Nat Genet 27:263-270, 2001 Downloaded from jco.ascopubs.org on April 29, 2010 . For personal use only. No other uses without permission. Copyright 2002 by the American Society of Clinical Oncology. All rights reserved.