Prognostic Value of Cytogenetic Findings in Adults With Acute Myeloid Leukemia

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1 Progress in Hematology International Journal of HEMATOLOGY Prognostic Value of Cytogenetic Findings in Adults With Acute Myeloid Leukemia Krzysztof Mrózek,* Kristiina Heinonen, Clara D. Bloomfield Division of Hematology and Oncology and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America Received June 19, 2000; accepted June 23, 2000 Abstract The majority of adults diagnosed with acute myeloid leukemia (AML) display acquired cytogenetic aberrations at presentation. Numerous recurring chromosomal abnormalities have been and continue to be identified in AML. In many instances, genes altered by these aberrations have been cloned, providing insights into the mechanisms of leukemogenesis and paving the way to designing novel therapeutic strategies that target specific genetic abnormalities in leukemic blasts. Moreover, karyotypic abnormalities, whether molecularly characterized or not, are among the most important independent prognostic factors in AML and are being used in the clinical management of AML patients. In this review, we present an overview of major cytogenetic findings in AML and discuss associations between karyotype and clinical outcome of adults with AML. Int J Hematol. 2000;72: The Japanese Society of Hematology Key words: Acute myeloid leukemia; chromosomal aberrations; karyotyping; prognosis 1. Introduction Cytogenetic studies of acute myeloid leukemia (AML) have contributed substantially to our understanding of the remarkable histopathologic, immunophenotypic, and clinical heterogeneity of AML. Multiple recurring chromosomal aberrations have been identified, and in many instances, genes altered by these aberrations have been mapped and cloned [1,2]. Further characterization of the genes rearranged by AML-associated translocations and inversions has provided insights into the mechanisms of leukemogenesis and will likely facilitate designing of novel therapeutic strategies that target particular genetic abnormalities in leukemic blasts [3,4]. In addition, acquired cytogenetic abnormalities, whether characterized at the molecular level or not, have been shown to represent tumor markers of diagnostic and prognostic importance [5,6]. Many recurrent aberrations have been correlated with presenting hematologic and morphologic parameters. Selected chromosomal aberrations, and their molecular equivalents, are now being used to help *Correspondence and reprint requests: Krzysztof Mrózek, MD, PhD, Division of Hematology and Oncology and the Comprehensive Cancer Center, The Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Rm. 1248B, The Ohio State University, 300 West Tenth Ave., Columbus, OH , USA; ; fax: ( mrozek-1@medctr.osu.edu). 261 define distinct disease categories within AML in the new World Health Organization classification of hematologic malignancies [7]. Moreover, karyotypic findings at diagnosis have been repeatedly shown to be among the most significant independent prognostic factors regarding AML [8-19]. In this article, we present major cytogenetic findings regarding AML and review their prognostic implications for adult patients with AML. 2. Overview of Cytogenetic Findings in AML Cytogenetic analysis is usually performed on a bone marrow or blood sample (preferably the former) that is obtained from an AML patient at diagnosis and contains leukemic blasts. The bone marrow sample is subjected to unstimulated 24- or 48-hour culture in vitro. Meaningful cytogenetic results are attained in the vast majority of adults with AML. Most large cytogenetic studies, involving more than 100 patients, have reported failure rates below 10% (range, 2% to 27%) [8,11-13,15,16,18-24]. Among successfully analyzed adults with AML, at least 1 clonal chromosomal aberration, ie, an identical structural rearrangement or an extra copy of the same chromosome present in at least 2 mitotic cells or the same chromosome missing from 3 metaphases, has been detected in 54% to 78% of patients [8,11-15,19,21-23,25-27], although both higher [24] and lower [16,28] percentages have also been reported. The rates of aberration detection in many [15,21,27,29] but not all [16,30] of more recent series have been higher

2 262 Mrózek et al / International Journal of Hematology 72 (2000) than those in the earliest studies [5]. This difference is likely because of improvements in cytogenetic methodology and because of the increased level of experience in detection of subtle structural aberrations, eg, inv(16)(p13q22), t(11;19) (q23;p13.1), or t(15;17)(q22;q12-21). Nonetheless, a substantial proportion of AML patients has an exclusively normal karyotype. The incidence of chromosomal abnormalities in AML may still be underestimated, as suggested by reports on karyotypically normal patients who are positive for gene fusion created by one of the recurrent chromosomal aberrations, eg, PML-RAR produced by t(15;17) or CBF - MYH11 by inv(16) [31-34]. Indeed, in rare cases, the presence of a chromosomal aberration may have escaped detection by the cytogenetic laboratory. In other patients, however, the gene fusion results from a cryptic rearrangement involving segments smaller than the length of a single band that is thus unrecognizable by standard cytogenetic analysis. Examples of cryptic insertions of a very small segment from 17q containing the RAR gene into the locus of the PML gene on chromosome 15q have been published [35]. On the other hand, the number of patients truly positive for gene fusion transcripts in the absence of cytogenetically detectable aberrations may be lower than indicated by some studies using reverse transcription polymerase chain reaction (RT-PCR) to detect rearrangements, because of the known propensity of RT-PCR to generate false-positive results [36,37]. At any rate, karyotypically normal AML patients positive for the specific gene fusion are rare and constitute only a fraction of all AML cases with a normal karyotype [23,31]. The notion that lack of microscopically discernible aberrations in a sizeable number of AML patients is an authentic phenomenon, not a failure in their detection, is supported by results of spectral karyotyping (SKY). SKY is a fluorescence in situ hybridization (FISH)-based technique allowing for simultaneous display of all human chromosomes in different colors [38]. SKY can potentially reveal cryptic interchromosomal abnormalities analogous to t(12;21)(p13;q22), which is a frequent chromosome rearrangement in pediatric B-lineage acute lymphoblastic leukemia. This translocation, arising from the juxtaposition of similarly banded regions, can be recognized microscopically only by FISH [1,4]. Recently, 2 series of 20 and 17 AML patients with normal karyotypes have been analyzed by SKY with no cryptic rearrangement found [39,40]. Thus, it is unlikely that aberrations undetectable by standard banding methods will be found by FISHbased techniques using chromosome painting probes in a large proportion of cytogenetically normal AML patients. Instead, leukemic blasts of some patients with a normal karyotype may harbor genetic abnormalities discernible only by molecular techniques such as RT-PCR or Southern blot analysis. For instance, approximately 11% of adults with de novo AML and a normal karyotype display rearrangements of the gene (also called ALL1, HRX, or Htrx1) detectable by Southern blot analysis [41,42]. In all cases with adequate material for additional molecular analysis, the gene rearrangement was a result of a partial tandem duplication (PTD) of a segment of the gene, with no evidence for involvement of another gene [42]. In another study, comprising patients with both de novo and secondary AML, the incidence of PTD was lower, 6% of cytogenetically normal patients [43]. Additional submicroscopic genetic changes in AML include dominant-negative mutation of the tumor suppressor gene C/EBP, reported in 24% of AML M2 patients with a normal karyotype [44], and point mutations in the CBFA2 (AML1 or PEBP2 B) gene [45]. It is plausible that other gene mutations contributing to leukemogenesis in karyotypically normal AML patients will be discovered in the future. Among the microscopically detectable chromosomal rearrangements, the more important ones appear to be those that are seen recurrently, can occur as the sole abnormality at least in some patients, or are rarely or never found in other types of hematologic and nonhematologic malignancies. These rearrangements are called primary aberrations and are presumed to play an important role in the early stages of leukemogenesis [5]. Table 1 lists those presumed primary abnormalities that have already been characterized molecularly.they are almost exclusively balanced rearrangements, reciprocal translocations, and inversions, which involve relocation of chromosomal segments between chromosomes or within a chromosome but do not lead to visible loss or gain of chromosomal material. At the DNA level, however, some such seemingly balanced aberrations may be more complex and involve multiple DNA breaks and further submicroscopic rearrangements of segments within the genes altered by a given translocation or inversion [46,47]. In addition to those listed in Table 1, several other balanced cytogenetic abnormalities have been detected recurrently in AML patients, but so far the genes affected by their creation are unknown. Three such reciprocal translocations, each reported in more than 10 patients, are presented in Table 2; the current list of other, less frequent AML-associated balanced rearrangements can be found in Mrózek et al [48]. It has been established that most reciprocal translocations and inversions in AML result in the fusion of genes that normally are involved directly or indirectly in the regulation of blood cell development [1,4]. Abnormal protein products of the fusion genes, which are frequently transcription factors, have been shown to be capable of dysregulating proliferation, differentiation, or apoptosis (programmed cell death) of blood cell precursors [1-4]. Similar effects may be brought about by loss or/and mutation of tumor suppressor genes (TSGs), which are presumed consequences of unbalanced aberrations that lead to the loss of a whole chromosome or a chromosomal segment [1,2,4,5]. To date, however, the actual TSGs affected by AML-associated deletions, isochromosomes, unbalanced translocations, and monosomies, the more common of which are presented in Table 2, have not been identified. The only exception is the TP53 gene found to be inactivated in many patients with AML (and with myelodysplastic syndrome [MDS]) harboring deletions or unbalanced translocations leading to the loss of 17p [49]. Alternatively, TSGs located in chromosomal regions rarely deleted in AML, eg, the cyclin-dependent kinase inhibitor p15 INK4B gene residing at 9p21, may also be inactivated with high frequency in both adult and childhood AML through an epigenetic mechanism, such as hypermethylation of CpG islands (i.e., regions of DNA rich in CpG dinucleotides) in the promoter region of the gene [50].

3 Prognostic Value of Cytogenetics in Adult AML 263 Table 1. Chromosomal Aberrations in Acute Myeloid Leukemia That Have Been Characterized at the Molecular Level* Aberration Gene Affecting the EVI1 gene at 3q26 inv(3)(q21q26) EVI1 t(3;3)(q21;q26), EVI1 Involving the NPM gene at 5q34 t(3;5)(q25;q34) MLF1-NPM Involving the MOZ gene at 8p11 inv(8)(p11q13) MOZ-TIF2 t(8;16)(p11;p13) MOZ-CBP t(8;22)(p11;q13), MOZ-EP300 Involving the nucleoporin genes CAN at 9q34 or NUP98 at 11p15 t(6;9)(p23;q34) DEK-CAN t(7;11)(p15;p15) HOXA9-NUP98 inv(11)(p15q22), NUP98-DDX10 t(11;20)(p15;q11) NUP98-TOP1 Involving the ABL gene at 9q34 t(9;22)(q34;q11)#,** ABL-BCR Involving the CLTH gene at 11q14 t(10;11)(p11-15;q13-23) AF10-CLTH Involving the gene at 11q23 t(1;11)(p32;q23),** AF1P- t(1;11)(q21;q23),** AF1Q- t(2;11)(p21;q23),** t(4;11)(q21;q23),** AF4- t(6;11)(q21;q23),** AF6q21- t(6;11)(q27;q23),** AF6- t(9;11)(p22;q23),** AF9- t(9;11)(q21-22;q23),** ins(10;11)(p11;q23q13-24),** t(10;11)(p11-13;q13-23),** AF10- t(10;11)(q22;q23),,** +11 t(11;11)(q13;q23),** t(11;15)(q23;q14-15),,** t(11;16)(q23;p13),** -CBP t(11;17)(q23;q12-21),** -AF17 t(11;17)(q23;q23),** t(11;17)(q23;q25),** -AF17q25 t(11;19)(q23;p13.1),** -ELL t(11;19)(q23;p13.3),** -ENL t(11;22)(q23;q11),,** -AF22 t(11;22)(q23;q13),,** -EP300 t(x;11)(q13;q23),** AFX1- t(x;11)(q22-24;q23),** Involving the ETV6 gene at 12p13 t(3;12)(q26;p13), ETV6 t(4;12)(q11-12;p13) BTL-ETV6 t(5;12)(q31;p13) ACS2-ETV6 t(7;12)(p15;p13), ETV6 t(7;12)(q36;p13), ETV6 t(12;13)(p13;q12) ETV6 t(12;22)(p12-13;q11-13), ETV6-MN1 Involving the core binding factor genes CBFβ at 16q22 or CBFA2 at 21q22 inv(16)(p13q22),** MYH11-CBF t(16;16)(p13;q22),** MYH11-CBF t(3;21)(q26;q22) EAP, MDS1,EVI1,CBFA2 t(8;21)(q22;q22), CBFA2T1-CBFA2 t(16;21)(q24;q22) MTG16-CBFA2 t(17;21)(q11.2;q22), CBFA2 Continued Table 1. Continued Aberration Gene Involving the RAR gene at 17q12-21 t(5;17)(q35;q12-21),,** NPM-RAR t(11;17)(q23;q12-21),** PLZF-RAR t(15;17)(q22;q12-21),** PML-RAR Involving the ERG gene at 21q22 t(16;21)(p11;q22) FUS-ERG *Data from reference 92. Additional data from references 72 and Chromosomal aberrations disrupting the same or related gene are grouped together. Within a given group, aberrations are arranged according to the numerical order of the first chromosome involved. Each aberration is presented only once. Reported in patients. Also interpreted as ins(3;3)(q21;q21q26). Reported in 6-25 patients. Chromosomal aberration reported in 5 patients or fewer. To date, not reported recurrently as a solitary aberration. #Reported in patients. **DNA probes for fluorescence in situ hybridization detecting rearrangements of genes (or 1 of the 2 genes) affected by a given aberration available commercially. Reported in >200 patients. Recurring 3-way variant translocations involving 5q31, 12q13, 17q23, and 20q13 also reported. In contrast to the balanced aberrations, the molecular mechanism whereby gain of an entire copy of a chromosome (trisomy) contributes to leukemogenesis is essentially unknown. To date, only trisomy 11 has been consistently associated with a specific molecular defect, in this case a PTD of the gene [51]. Although as many as 55% of cytogenetically aberrant AML patients have only 1 chromosomal abnormality in their marrow karyotype [5], such an aberration is not the sole genetic alteration sufficient for transformation of a normal stem cell into a leukemic blast. Malignant transformation is a multistep process involving accumulation of several genetic rearrangements, both those detectable by cytogenetic analysis (primary aberrations and, accompanying them, secondary chromosomal changes) and those discernible by molecular genetic methods only, as well as epigenetic events such as hypermethylation of the regulatory regions of TSGs resulting in their inactivation [4]. This process has been demonstrated in a recent study in mice, which has provided experimental evidence that the Cbf-MYH11 fusion gene can block myeloid differentiation and predispose to leukemia. The gene does not instigate leukemogenesis by itself, however; the acquisition of additional, not yet known, mutations is required [52]. 3. Prognostic Relevance of Cytogenetic Findings in AML It has now been well established that results of cytogenetic analysis at diagnosis provide important prognostic information in AML. It is worthy to note that although both molecular genetic methods (RT-PCR, Southern blot analysis) and FISH are now widely available for detection of

4 264 Mrózek et al / International Journal of Hematology 72 (2000) many recurrent chromosomal aberrations in AML [1,4,37], the vast majority of data correlating genetic features of leukemia with the clinical outcome have been obtained from studies using standard cytogenetic analysis as the primary investigative tool. Thus, with the exception of t(15;17)/pml- RAR -positive acute promyelocytic leukemia (APL), prospective investigations need to test whether conclusions reached by clinical-cytogenetic studies hold up if only molecular techniques are used for detection of chromosome changes. This future testing is especially important in light of significant discrepancies between results of cytogenetic and RT-PCR analyses in some recent retrospective series [33,53]. Likewise, the clinical applicability of emerging microchip array technology, which will likely make possible rapid detection of all molecularly characterized genetic rearrangements in AML simultaneously [37], will have to be verified before it can become part of the management of patients with AML. The significance of the karyotype as an independent determinant of outcome was demonstrated conclusively for the first time in a large, prospective, multicenter study at the Fourth International Workshop on Chromosomes in Leukemia (IWCL) [8]. This study and its first follow-up, the Sixth IWCL [9], corroborated earlier observations that AML patients with a normal karyotype (designated NN) were more likely to suvive longer than patients with a mixture of abnormal and normal (residual) mitotic cells (AN) or those with abnormal Table 2. More Common Chromosomal Aberrations in Acute Myeloid Leukemia Not Yet Characterized Molecularly* Balanced aberrations t(1;3)(p36;q21) t(1;22)(p13;q13), t(3;5)(q21;q31) Unbalanced chromosomal rearrangements Resulting in loss of a chromosome or chromosomal segment del(1)(q21) del(2)(p21-23) 5 del(5)(q12-31-q31-35),# del(6)(q13-24-q21-27) 7 del(7)(q11-34-q22-36),** 9 del(9)(p21) del(9)(q11-22-q21-34), del(10)(p12) del(11)(p11-12-p14-15) del(11)(q13-23-q22-25), del(12)(p11-13) del(13)(q11-22-q14-34) del(16)(q21-22-q24) del(17)(p11-13) del(20)(q11-13), 21 Y Resulting in simultaneous loss and gain of a chromosomal segment der(1;7)(q10;p10), i(7)(q10) Continued Table 2. Continued i(11)(q10) i(13)(q10) i(14)(q10) i(17)(q10) i(21)(q10) idic(x)(q13) Resulting in gain of a chromosome or chromosomal segment +i(1)(q10) i(12)(p10) *Data from references 5, 92, and 99. Only aberrations reported as solitary chromosomal changes in at least 2 patients with AML are included. Within a given category, abnormalities are arranged according to the numerical order of the chromosome(s) involved. Chromosomal aberration reported in 6-25 patients. For numerical aberrations (monosomies and trisomies), footnotes,,, and indicate the numbers of patients harboring a particular aberration as a sole abnormality only; for structural aberrations, the numbers provided refer to all patients, irrespective of whether a given aberration is isolated or not. Thus far detected exclusively in children, mostly with AML M7. Reported in patients. (See for numbers of patients harboring a particular aberration.) Although many deletions are reported as terminal, they are generally regarded as being interstitial. Reported in >200 patients. (See for numbers of patients harboring a particular aberration.) #del(5)(q13q33) the most common aberration. **del(7)(q22) the most common aberration. Reported in patients. (See for numbers of patients harboring a particular aberration.) del(9)(q22) the most common aberration. del(11)(q23) the most common aberration. del(20)(q11) the most common aberration. Reported also as der(1)t(1;7)(p11;p11). cells only (AA). However, there was no significant difference in survival between AN and AA patients [9], and the probability of their achieving complete remission (CR) was not dependent on whether normal metaphases were present [8]. In the multivariate analysis of survival, when the other main risk factors in AML such as age, sex, and French-American- British classification subtype were also considered, the NN-AN-AA classification was found to have an independent prognostic significance in both the group of 656 patients with de novo AML and the subset of 305 patients who received more intensive induction treatment with cytarabine and an anthracycline [9]. Analogous results have been obtained in 2 other studies of adults with de novo AML [11,15]. However, 1 group has not confirmed the prognostic value of the NN-AN- AA classification, perhaps because more than one-half of patients in their AA category had the prognostically favorable t(8;21) [28].

5 Prognostic Value of Cytogenetics in Adult AML 265 It appears that prognostic relevance of the presence or absence of residual, normal metaphase cells in karyotypically aberrant AML patients depends on the kind of chromosomal aberrations found in leukemic blasts. Ghaddar et al [54] reported no difference in CR rate, CR duration (CRD), and survival between AN and AA patients who had either inv(16) or t(8;21). There was also no difference in CRD between AN and AA patients with t(15;17). The latter finding was confirmed in a series of t(15;17)-positive APL patients treated by chemotherapy alone, without all-trans-retinoic acid (ATRA), in which no difference in CR rate, event-free survival, and overall survival between AN and AA patients was found [55]. In contrast, when AML patients with 5, del(5q), 7, del(7q), or +8 at diagnosis were combined into 1 subset, the clinical outcome of AN patients was significantly better than that of AA patients who did not have any normal mitoses [54]. Another system for classifying karyotypes, the complexity classification, was also found at the Sixth IWCL to represent an independent prognostic factor [9]. Patients with very complex karyotypes ie, when more than 5 chromosomes were rearranged in the abnormal clone had the shortest survival, whereas those with either normal karyotypes or an abnormal clone or clones involving 2 to 5 chromosomes survived longest [9]. In a smaller study, AML patients with a complex karyotype, specified as containing 3 or more clonal chromosomal aberrations, had a significantly lower CR rate and shorter survival than those of patients with a normal karyotype or patients with inv(16), t(8;21), t(15;17), or 1 or 2 other aberrations [16]. A complex karyotype, also defined as containing at least 3 aberrations, was predictive of shorter survival in a group of patients aged 56 years or older, but not in patients younger than 56 years, in a series of more than 200 adults with de novo AML [26]. On the other hand, in the largest cytogenetic study reported to date, comprising 1612 AML patients younger than 56 years, including 340 children, complex karyotypes containing at least 5 unrelated cytogenetic abnormalities were associated with a CR rate significantly lower, relapse risk significantly higher, and overall survival significantly shorter than those of patients with a normal karyotype [18]. Although the aforementioned general systems for classifying cytogenetic results may be useful in some groups of patients with AML, the most important prognostic information is gained from detection of specific chromosomal aberrations. At the Fourth IWCL, significant differences in CR rate, CRD, and overall survival time were shown when the 716 patients were classified by karyotype in a prioritized schema, first according to the presence of t(8;21); then t(15;17), 5 or del(5q), 7 or del(7q); simultaneous presence of 5 or del(5q) and 7 or del(7q); followed by abnormalities of 11q, +8, and +21. The remaining patients were classified according to the ploidy level (hypodiploid, pseudodiploid, diploid [normal], and hyperdiploid). Karyotypes were independent prognostic factors for duration of first CR and of overall survival in the group of 305 adequately treated patients [8]. In the follow-up studies of the Fourth IWCL, a group comprising patients with inv(16)(p13q22) and del(16)(q22) was added, all cases with numerical and/or structural abnormalities of chromosomes 5 and 7 were combined into a single group, and patients with +8 and +21 were included in the hyperdiploid group [8-10,56].The multivariate analyses performed at the third follow-up of the Fourth IWCL, which then comprised 628 patients with primary AML and a median follow-up of 14.7 years for living patients, confirmed that karyotype remained an independent predictor of survival for all patients and for the 291 patients who received induction therapy considered as standard by modern criteria [10]. Similarly, other studies in which specific cytogenetic findings were categorized in various ways have confirmed that karyotype represents an independent prognostic determinant for attainment of CR [11-13,15,19], for CRD [11,15], and for overall survival time [14-16,57]. In most studies of adults with de novo AML, the highest CR rates, the longest CRD, and overall survival time have been associated with t(8;21) and inv(16) (Table 3). Both of these chromosomal aberrations are related at the molecular level because t(8;21) disrupts a gene encoding the subunit and inv(16) disrupts a gene encoding the subunit of corebinding factor (CBF). CBF is a transcription factor involved in regulating a number of genes involved in hematopoiesis and is necessary for normal development of the hematopoietic system [58]. The chimeric proteins encoded by fusion genes CBFA2T1-CBFA2 and MYH11-CBF are both able to repress CBF-mediated transcriptional activation of target genes in a dominant fashion [58], and this ability may trigger a common leukemogenic pathway. The blasts carrying either t(8;21) or inv(16) appear to be more sensitive to currently used treatment regimens than blasts with other aberrations, although the molecular basis of a good response to chemotherapy of patients with CBF leukemia has not yet been elucidated. Their superior response may arise from an enhanced sensitivity of the blasts to cytarabine, which, together with anthracyclines, constitutes a mainstay of current chemotherapy for AML. A significant increase in in vitro incorporation of cytarabine into nuclear DNA and cytarabine-induced apoptosis of cells from patients with inv(16) compared with blasts from patients with other chromosomal rearrangements or a normal karyotype has been recently reported [59]. It has been demonstrated that intensive postremission therapy with high-dose cytarabine (HDAC) in adults with de novo AML and t(8;21), inv(16)/t(16;16) or a normal karyotype, but not in those with other aberrations, improves their outcome substantially [17]. The HDAC intensification treatment is especially effective in patients with t(8;21).as shown by the third follow-up of the Fourth IWCL, no patient with t(8;21) who attained a CR survived continually disease-free for 10 years in the absence of HDAC intensification. In contrast, 89% of complete responders who were administered HDAC on the Cancer and Leukemia Group B (CALGB) 8525 protocol were estimated to be cured using a Farewell Mixture Model [10]. Another CALGB study has indicated that t(8;21)-positive patients who receive the highest cumulative dose of cytarabine in the course of intensification treatment benefit the most [60]. The projected 5-year disease-free survival (71% versus 37%) and overall survival (76% versus 44%) rates were superior in patients who were given 3 or 4 cycles of HDAC compared with those who received 1 cycle only of HDAC followed by sequential treatments with cyclophosphamide/etoposide and mitoxantrone/diaziquone with or without filgrastim [60]. Notably, the patients assigned

6 266 Mrózek et al / International Journal of Hematology 72 (2000) to receive 3 or 4 cycles of HDAC did not differ from those assigned to receive only 1 cycle with regard to pretreatment factors previously reported to confer worse prognosis in patients with t(8;21), such as absolute granulocyte count greater than /L, leukocyte count more than /L, the presence of granulocytic sarcoma, expression of the neural cell adhesion molecule CD56 on leukemic blasts, or secondary chromosomal aberrations additional to t(8;21), including del(9q) and loss of chromosome Y in males or chromosome X in females [60]. Among patients with inv(16)/t(16;16), treatment with HDAC appears to lower the risk of development of intracerebral myeloblastoma [61], thus making prophylactic central nervous system irradiation or intrathecal chemotherapy unnecessary. Nevertheless, despite the generally good prognosis of adults positive for inv(16) and its improvement by HDAC, not all such patients are cured. One relatively small study suggested that treatment results are better in patients who in addition to inv(16) harbor a submicroscopic deletion of the gene for multidrug resistance associated protein (MRP), located proximally to the short-arm breakpoint of the inversion, at 16p13.1 [62]. However, 3 subsequent studies [63-65], including the most recent one in which the patients received uniform induction and consolidation treatment with HDAC and mitoxantrone [65], have not confirmed the favorable influence of MRP gene deletions on prognosis. Additionally, the treatment outcome of patients with inv(16)/t(16;16) does not seem to be affected by the presence of various secondary aberrations [18,66-68] that may accompany inv(16) in more than 40% of patients [69]. Thus, other factors are likely responsible for the clinical heterogeneity within this subset of AML. Table 3. Associations Between Selected Recurrent Chromosomal Findings and Clinical Outcome in Adults With De Novo Acute Myeloid Leukemia* Complete Remission Survival Chromosome Aberration Rate, % (n) Median, mo Probability, % (y) Median, mo Probability, % (y) t(8;21)(q22;q22) A 93 (563) ~16 (3) to 71 (5) to 69 (5) t(16;21)(p11;q22) B 90 (20) NA NA 13 NA inv(16)(p13q22) C 87 (367) ~33 (3) to 62 (5) (4) to ~76 (5) t(6;11)(q27;q23) D 85 (26) 8 NA 12 4 (2) t(9;11)(p22;q23) E 79 (24) (5) (5) t(11;19)(q23;p13.1) F 79 (19) NA NA NA 26 (2) t(15;17)(q22;q12-21) G 78 (559) ~15 (3) to 63 (5) 8-33 ~8 (3) to 63 (5) None H 76 (1929) (5) 7-31 ~7 (3) to 42 (5) del(7q) J 75 (32) NA 41 (5) NA 23 (5) +8 (sole) K 73 (134) (3) to 33 (5) (3) to 27 (5) Abnormalities of 11q23 L, 63 (125) (3) to 33 (5) (3) to 11 (5) +11 (sole) M 61 (38) (4) (4) del(5q) N 57 (28) NA 15 (5) NA 11 (5) 7 O 56 (66) NA 20 (5) NA 10 (5) +8 P # 53 (88) 8-24 NA (5) to 19 (3) Abnormalities of 12p R 47 (38) NA NA NA 11 (2) del(20q) or 20 S 46 (11) 7 NA 9 17 (3) +13 (sole) T 43 (40) NA NA 3 NA 5 U 42 (26) NA 10 (5) NA 4 (5) Unbalanced abnormalities of 11q23 V, ** 41 (29) 5-6 NA 2 NA t(9;22)(q34;q11) W 38 (42) 6 NA 7 NA inv(3)(q21q26)/t(3;3)(q21;q26) X 30 (27) 6 NA 9 0 (3) *Some studies also included 15% to 18% of patients diagnosed with MDS [79,120], or 5% to 37% of patients with secondary acute myeloid leukemia (ie, following an antecedent hematologic disorder or chemo- or radiotherapy for prior malignancy) [11,18,27,67,68,79,104,109,111,114], or 2% to 21% of children with AML [12,13,18,21,86,101,103,104,106, ]. Aberrations are arranged according to the associated average complete remission (CR) rate (from the highest to the lowest), calculated from data on all patients with a given aberration reported in the studies referenced. A superscript capital letter placed after a given abnormality denotes reference numbers identifying articles containing clinical outcome data on patients with this aberration, as follows: A [11,12,18,21,25-28, ], B [101,102], C [12,18,21,25-27,66-68,86,105,109], D [110], E [78], F [111], G [11-13,15,18,21,25-27,55,86,112,113], H [11-13,15,16,18,21,25-27,105], J [18], K [18,26,28,84-86], L [12,13,15,25-27,78,79,114], M [21,28,51,87,115,116], N [18], O [13,18], P [11-13,15,16,27], R [117], S [27,118], T [119,120], U [18], V [114,121], W [11,12,21,26-28,122,123], X [27,74,124,125]. For a given aberration, only the lowest and the highest probability of remaining in CR or surviving, respectively, are provided. ~ denotes data obtained from Kaplan-Meier plots in reference. Numbers in parentheses indicate the time points in years of when the probability was determined. NA, data not available. Actual percentage of patients alive at a given time point. In most studies, this group contains all types of translocations involving band 11q23, including t(9;11)(p22;q23), and deletions of 11q23, except in Mrózek et al [78], where t(9;11) and del(11q) have been excluded. There is no clear distinction between 7 as a sole abnormality and as a secondary change accompanying other aberrations. #There is no clear distinction between +8 as a sole abnormality and as a secondary change. **Includes 16 cases with del(11q) and 13 cases with unbalanced translocations with known or unknown partner chromosomes. In reference 74, the outcome data provided are for a group comprising 7 patients with t(3;3)/inv(3) and 1 patient with t(3;12)(q26;p13).

7 Prognostic Value of Cytogenetics in Adult AML 267 Patients with t(15;17) represent another group characterized by unique morphologic features and presenting clinical features and a good prognosis. The average CR rate for adults with t(15;17) in Table 3 is only 78%, but this has been calculated from studies that predominantly involved APL patients treated with chemotherapy alone, without ATRA. The addition of ATRA to the induction regimen has been reported to raise the CR rate in such patients to between 85% and 91%, and to significantly prolong disease-free and overall survival [32,70]. The importance of detecting t(15;17) in all APL patients is underscored by results of a recent intergroup study in which none of the 5 patients considered to have APL-like marrow morphology, but who were PML-RAR -negative (and hence did not have the t[15;17]), achieved a CR when treated with ATRA [71]. Correspondingly, patients with APL-like AML and t(11;17) (q23;q21) are not responsive to ATRA even at high doses; they also have a poor prognosis regardless of whether chemotherapy or ATRA is used as induction therapy [72]. Adults with an entirely normal karyotype seem to have an intermediate prognosis. Their CRD and survival are shorter than those of adequately treated patients with t(8;21), inv(16), or t(15;17) but are longer than patients with unfavorable chromosomal aberrations (Table 3). However, this group is highly heterogeneous and is likely composed of subsets with varying prognoses. For instance, the presence of a submicroscopic PTD of the gene identifies a subgroup of karyotypically normal patients characterized by a significantly shorter CRD [42,43] and overall survival [43] than patients with a normal karyotype and the germline. Another feature associated with worse prognosis of adults with de novo AML and a normal karyotype may be the presence of erythroblastic and/or megakaryoblastic dysplasia (EMD) in the diagnostic marrow. In a relatively small study, cytogenetically normal patients with EMD had a significantly lower CR rate and a shorter event-free and overall survival than did similar patients without EMD [73]. Future studies will likely discover other factors, including submicroscopic gene mutations, that can affect the treatment outcome of AML patients with normal cytogenetics. A number of chromosomal aberrations have been repeatedly associated with a poor prognosis. These include inv(3) (q21q26) or t(3;3)(q21;q26), del(5q) or 5, 7, t(9;22) (q34;q11), and abnormalities of 12p. Prolonged CRD and long survival are uncommon in patients with these aberrations (Table 3). Of note, in the case of inv(3) and t(3;3), it is the presence of microscopically identifiable chromosomal aberrations, not inappropriate overexpression of the EVI1 gene, detectable by RT-PCR in both patients with inv(3)/t(3;3) and those without these aberrations, that has adverse prognostic significance [74]. Deletions of 7q occurring concurrently with del(5q) or 5 or as part of a complex karyotype have also been associated with poor prognosis. In contrast, the outcome of patients with del(7q) in the absence of unfavorable cytogenetic features, ie, a complex karyotype, abnormalities of 3q or del(5q)/ 5, did not differ significantly from that of patients with a normal karyotype in a study reported by Grimwade et al [12]. Their findings are consistent with earlier observations that patients with del(7q) without concurrent aberrations of chromosome 5 may have prolonged survival [26,56]. A recent study indicates that prognosis of AML/MDS patients with 5/del(5q) and/or 7/del(7q) may also be influenced by the stability of the leukemic karyotype. Estey et al [75] analyzed treatment outcome of 400 patients with the aforementioned aberrations [excluding patients with concomitant inv(16) or t(8;21)] who were diagnosed with de novo AML, AML secondary to an antecedent hematologic disorder (AHD), refractory anemia with excess of blasts, or refractory anemia with excess of blasts in transformation. In general, the clinical outcome of this group was poor, with a median survival of 4 months. However, although the CR rates were the same (40%), the disease-free survival once in CR and overall survival were significantly longer for patients with only 1 abnormal clone versus those with the less stable karyotype containing 2 or more clones. By using 2 other factors to stratify the patients, ie, presence or absence of residual normal cells and presence or absence of AHD, Estey et al recognized a small (10%) subset of patients with 5/del(5q) and/or 7/del(7q) with a better prognosis, equivalent to that of similarly treated patients with a normal karyotype. Patients in this subset had only 1 abnormal clone, at least 1 normal mitotic cell, and no history of AHD [75]. These results should be corroborated in more homogeneous patient populations. Patients with other recurrent aberrations have been variously placed in either the intermediate [18,57,76] or unfavorable [10,17,77] prognostic groups. This distinction, to a certain extent, reflects the infrequency of many recurrent abnormalities in AML that has thus far precluded reliable assessment of their prognostic importance. Furthermore, certain cytogenetic categories may be heterogeneous. Once an adequate number of uniformly treated patients are studied, they may be further divided into subgroups with disparate prognoses. For instance, in our study of treatment outcome in adults with de novo AML and balanced abnormalities involving band 11q23 [78], patients with t(9;11) (p22;q23), who in most earlier studies had been grouped together with patients with other 11q23 aberrations [11,12, 15,25-27,79], had a significantly longer CRD, event-free survival, and overall survival than adults with translocations between 11q23 and partner chromosomes other than 9p22 [78]. Likewise, patients with t(9;11) had a better outcome than those with t(10;11)(p12;q23) in another study, although the difference was not statistically significant [18]. In most [80,81] but not all [82] series of childhood AML, t(9;11) conferred a significantly better prognosis than other types of 11q23 anomalies. Thus, regardless of age, detection of the gene rearrangement by Southern blot analysis alone, without identification of the 11q23 partner chromosome by standard cytogenetic analysis, FISH, or RT-PCR, is not sufficient for predicting the patients clinical behavior [78]. Notably, the prognosis of adults with t(9;11) does not seem to be unfavorably influenced by the presence of +8 as a secondary aberration [78], nor does it correlate with immunophenotypic features of leukemic cells, including lymphoid antigen expression [83]. Our preliminary findings suggest that patients with de novo AML and t(9;11), but not those with other 11q23 translocations, may particularly benefit from intensive postremission therapy in first CR [78]. The efficacy

8 268 Mrózek et al / International Journal of Hematology 72 (2000) of intensification regimens containing HDAC alone or in combination with other drugs, including etoposide, shown to be especially effective in children with myelomonocytic and monocytic leukemias, should be assessed prospectively in adults with t(9;11) and, if confirmed, might become the therapy of choice for patients when stem cell transplantation is not available [78]. Substantial variation in CR rates (from 29% [12] to 91% [26]), CRD, and survival rates has been observed among AML patients with +8 (Table 3). In some reports [11-13,15,16,27], this cytogenetic group included both patients with isolated +8 and those who in addition to +8 had other aberrations that may have affected response to treatment and outcome. Selective detection of +8 by interphase FISH is not adequate to determine prognosis, as has been shown by recent studies reporting large differences in outcome between patients with solitary +8 and those in whom +8 occurred in addition to aberrations conferring either favorable or adverse prognoses [18,84]. However, the differences in outcome among studies are also notable when patients with isolated +8 only were included [18,26,84-86]. This variability could be associated with differences in the age of the patients analyzed by different groups. Byrd et al [85] have shown that the CR rate of patients older than 60 years with solitary +8 is significantly lower than that of younger patients (40% versus 88%; P =.004), as is their overall survival (median, 4.8 months versus 17.5 months; P =.01). Patients younger than 35 years, including a substantial number of children, dominated among patients with +8 in the study of Grimwade et al [18], who reported a relatively good outcome for patients with isolated +8. The adverse effect of older age on treatment outcome was also observed in adults with isolated +11 [87] and in patients constituting the poor prognosis group [ie, patients with der(1;7)(q10;p10), inv(3), del(5q)/-5, 7, t(9;22), or complex karyotypes] and the intermediate prognosis group [ie, patients with a normal karyotype or aberrations other than the aforementioned unfavorable ones or favorable t(8;21), inv(16), and t(15;17)] in a population-based series of 372 adults with AML [76]. On the other hand, a relatively good prognosis for patients belonging to a favorable group did not deteriorate with age, and the median survival was even longer in the group of oldest patients (65 to 74 years) than in patients aged 50 to 64 and 20 to 49 years [76]. 4. Conclusions The prognostic significance of several of the more frequent karyotypic aberrations has now been well established. Recently, cytogenetic findings have been integrated into a prognostic index applicable in risk-directed therapy decisionmaking for younger patients with AML [57]. However, many less common aberrations, whose individual impact on the clinical outcome is at present unknown, have been combined into 1 prognostic category, which may be an oversimplification. Moreover, novel chromosomal aberrations are still being discovered in AML patients, using both standard cytogenetic analysis and molecular-cytogenetic techniques such as FISH, SKY, and rainbow cross-species FISH (Rx-FISH) [39,88-90]. Accurate detection of genetic abnormalities becomes even more important as therapeutic agents designed to target specific molecular rearrangements associated with recurrent chromosomal aberrations in AML become available. Such an agent, STI 571, a protein tyrosine kinase inhibitor that suppresses proliferation of cells harboring the BCR-ABL fusion gene created by t(9;22), has been successfully used to treat patients with chronic myelogenous leukemia who have failed interferon therapy [91]. It is well known that prognostic factors in AML depend on the type of induction and, perhaps to an even greater extent, postremission treatment used [10,17,60]. An abnormality conferring an adverse prognosis with 1 therapeutic regimen may lose its unfavorable prognostic impact when another treatment is used. Hence, a clear need exists for large prospective studies evaluating associations between karyotype and clinical outcome. Such studies will facilitate evaluation of the prognostic impact of the less frequent chromosomal aberrations, both primary and secondary, whose clinical significance is not yet determined. 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