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FLT3 mutational studies on 17 patients with normal karyotype acute myeloid leukemia (AML) followed by aberrant karyotype AML at relapse
Cancer Genetics and Cytogenetics 202 (2010) 101e107
Genomic, immunophenotypic, and NPM1/FLT3 mutational studies on 17 patients with normal karyotype acute myeloid leukemia (AML) followed by aberrant karyotype AML at relapse Eunice S. Wanga,*, Sheila N.J. Saitb, David Goldc,d, Terry Mashtarec,d, Petr Starostikb, Laurie Ann Forda, Meir Wetzlera, Norma J. Nowake, George Deebb a Leukemia Service, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 c Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, NY 14263 d Department of Biostatistics, SUNY-UB School of Public Health, Buffalo, NY 14214 e Department of Cancer Prevention, Roswell Park Cancer Institute, Buffalo, NY 14263 Received 23 February 2010; received in revised form 28 June 2010; accepted 2 July 2010
b
Abstract
Normal karyotype (NK) is the most common cytogenetic group in acute myeloid leukemia (AML) diagnosis; however, up to 50% of these patients at relapse will have aberrant karyotype (AK) AML. To determine the etiology of relapsed AK AML cells, we evaluated cytogenetic, immunophenotypic, and molecular results of 17 patients with diagnostic NK AML and relapsed AK AML at our institute. AK AML karyotype was diverse, involving no favorable and largely (8 of 17) complex cytogenetics. Despite clear cytogenetic differences, immunophenotype and NPM1/FLT3 gene mutation status did not change between presentation and relapse in 83% (10 of 12) and 94% (15 of 16) cases, respectively. High-resolution array-based comparative genomic hybridization (aCGH) performed via paired aCGH on NK AML and AK AML samples from the same patient confirmed cytogenetic aberrations only in the relapse sample. Analysis of 16 additional diagnostic NK AML samples revealed no evidence of submicroscopic aberrations undetected by conventional cytogenetics in any case. These results favor evolution of NK AML leukemia cells with acquisition of novel genetic changes as the most common etiology of AK AML relapse as opposed to secondary leukemogenesis. Additional studies are needed to confirm whether AK AML cells represent selection of rare preexisting clones below aCGH detection and to further characterize the molecular lesions found at time of AK AML relapse. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Acute myeloid leukemia (AML) is a rapidly proliferating neoplasm of immature hematopoietic cells. Diagnostic karyotype is well established as the best independent prognostic factor for AML for complete remission as well as for disease-free and overall survival. Normal karyotype (NK) AML is the most common AML cytogenetic group, comprising 40e49% of adult and 20e25% of pediatric AML diagnoses. NK AML is associated with an intermediate prognosis, defined as a 5-year survival rate of 24e40% [1,2]. Despite the importance of diagnostic karyotype [3], however, 25e51% of NK AML patients will have * Corresponding author. Tel.: (716) 845-3544; fax: (716) 845-8741. E-mail address: [email protected] (E.S. Wang). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.07.117
AML associated with aberrant karyotype (AK) at relapse [4e7]. It is unknown how these previously cytogenetically normal AML cells “acquire” additional genetic abnormalities [5]. One possibility is that AK AML cells were present but undetected at time of NK AML diagnosis as a result of low cell frequency and the relative insensitivity of conventional cytogenetic analyses. Recent analyses have shown that most relapsed pediatric acute lymphocytic leukemia (ALL) disease arises from minor tumor subpopulations present but undetected at time of diagnosis [8]. Alternatively, AK AML cells could have arisen from acquired genetic changes to preexisting NK AML cells. Finally, AK AML cells may represent secondary de novo leukemia cells. To elucidate the etiology of AK AML after NK AML, we identified 17 patients with diagnostic NK AML and
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relapsed AK AML (designated NK-AK AML) treated at our institute. We compared cytogenetic and immunophenotypic profiles, and NPM1/FLT3 gene mutation status from patient samples at diagnosis and relapse. In addition, we performed high-resolution array-based comparative genomic hybridization (aCGH) on all diagnostic NK AML samples specifically looking for chromosomal aberrations that might have been present but undetected by conventional karyotyping [9e11].
2. Materials and methods 2.1. Patient samples Under an IRB-approved protocol, we reviewed data on 301 consecutive NK AML patients diagnosed at our institute between 1991 and 2009. Clinical outcomes were defined per Cheson et al. [12]. Relapse was defined as the finding of O5% marrow blasts not attributable to another cause or to extramedullary disease. Event-free survival was defined as time between relapse and death from any cause. Sixty patients had karyotype information at both diagnosis and time of disease progression and/or relapse. Of these, 30 had NK AML at diagnosis and AK AML at relapse (NK-AK AML), and 30 had NK AML at both diagnosis and relapse (NK-NK AML). Seventeen NK-AK AML patients diagnosed between December 2002 and April 2008 were selected for further analysis on the basis of availability of stored cells at NK AML diagnosis containing 45% blast cells, which is considered the minimum unsorted cell threshold to undergo aCGH analysis. Because all 17 NK AML cases lacked recurrent genetic abnormalities recognized by World Health Organization classification, they were classified morphologically according to FrencheAmericaneBritish classification. Multiparameter four-color flow cytometry [13] was reviewed on all available diagnostic and relapsed AML samples. Genomic DNA from fresh or cryopreserved NK AML marrow aspirate cells was also analyzed for FLT3 and NPM1 mutations. FLT3 gene internal tandem duplications and tyrosine kinase domain mutations were performed by polymerase chain reaction (PCR) methods as previously described [14]. NPM1 mutational analysis was performed by PCR amplification of insertions occurring at the 50 intron/exon boundary followed by separation/sizing by capillary electrophoresis on an ABI 3130xl Genetic Analyzer. Both forward and reverse PCR primers were hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxyfluorescein (FAM) labeled, respectively. A wild-type result consisted of HEX- and FAM-labeled PCR products at 122 bp and 125 bp, respectively. NPM1 mutants yielded FAM- and HEX-labeled PCR products that were greater in size than wild type. Sequences were as follows: NPM1 F fam2 5 50 -6-FAM-TTT-CTT-TTT-TTT-TTT-TTC-CAGGC -30 ; NPM1R3 hex 5 50 -HEX-AGA-TAT-CAA-CTGTTA-CAG-AAA-TGA-AAT-AAG-30 .
2.2. Array-based comparative genomic hybridization All patient samples were procured after informed patient consent was obtained under an institutional review board (IRB) protocol. aCGH was performed under a separate approved IRB protocol. Genomic DNA was prepared using FlexiGene DNA Isolation kit (Qiagen, Valencia, CA) from O1e2 106 unsorted AML patient marrow mononuclear cells. aCGH utilizing 19K bacterial artificial chromosomes (RP11 bacterial artificial chromosome [BAC] library) with image analysis was performed [15e17]. Genomic aberrations were identified by Nexus software, version 3.0 (BioDiscovery, El Segundo, CA), with a built-in Rank Segmentation algorithm and a cutoff of þ0.15/0.15 based on prior studies of human leukemia samples [18,19]. All cases showed chromosome X and Y gain/loss signals compatible with the patient’s sex based on this cutoff and were compared with sex-matched normal controls. Statistical significance of copy gains/ deletions identified by Nexus was inferred with CGHcall algorithm, using an 80% threshold on the conditional posterior probability in favor of gain or deletion, respectively [20].
3. Results 3.1. Clinical characteristics Characteristics of the 17 NK-AK AML patients evaluated here are summarized in Table 1. All patients received induction therapy with cytarabine- and anthracycline-based induction AML regimens before relapse or recurrence. Overall time and time to AK AML development were 470 213 days and 294 198 days, respectively. Eventfree survival (or survival after AK AML diagnosis) was only 96 64 days. Table 1 NK-AK AML patient characteristicsa Characteristic
Value
Age (y), range (median) Sex (M/F) FAB classification WBC (109/L) Diagnosis Relapse Blasts (%) Diagnosis Relapse Overall survival (d) Time to AK AML (d) Event-free survival (d)
68 (21e81) 9/8 7 M1, 7M2, 2 M4, 1 M5a 37.7 39 (2.01e138.1) 4.76 53 (0.25e192.1) 80 16 (48e95) 73.5 23 (23e97) 470 213 (240e1,110) 294 198 (105e986) 96 64 (18e202)
Abbreviations: NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; FAB, FrencheAmericaneBritish; WBC, white blood cell count. a Data are expressed as median standard deviation (range) unless indicated otherwise.
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Table 2 Cytogenetic abnormalities detected by aCGH and karyotype in NK-AK AML samplesa Patient no.
Diagnosis karyotype
Diagnosis aCGH analysis (band/gain or loss)
02-1407 04-0391
46,XX[20] 46,XY[13]
5q13.2 gain (NS, CNV rich) 21p11.1 loss (NS)
03-0875
46,XY[20]
04-1272 06-0130
46,XY[20] 46,XX[20]
11q22.3 gain (NS) 16p11.2e11.1 gain (NS) 21p11.1 loss (NS) No gains/losses 21 p11.2e11.1 gain (NS) 9p13.3e11.2 loss (S, PC)
05-1177 05-1517
46,XX[25] 46,XX[20]
1q31.1e32.1 (NS) No gains/losses
04-0714
46,XX[21]
1q21.1e21.2 loss (NS)
04-0967 04-0071
46,XY[19] 46,XY[16]
9p13.3e11.2 loss (S, PC) No gains/losses
03-0106 05-0210
46,XX[25] 46,XX[20]
21p11.2e11.1 gain (NS, PC) 21p11.2e11.1 gain (NS) (PC)
06-0614 06-0394
46,XY[20] 46,XX[20]
No gains/losses No gains/losses
03-1152 07-0924
46,XY[20] 46,XY[20]
08-0450
46,XY[20]
9p13.1e11.2 loss (S, PC) 1q12e21.1 gain (NS, PC, CNV-rich) 21p11.2e11.1 gain (NS) 7p21.1 loss (NS)
Relapse karyotype 46,XX,del(11)(p11.2p14)[19] 46,XY,t(11;16)(p13;q22)[5] 46,XY,t(9;11)(p13;q13),t(12;22)(q13;q13)[9] 46,XY,t(13;17)(q14;q11.2)[14]
46,XY,t(2;3)(p21;p21),del(3)(q21q26),t(7;11)(p21;p14)[19] 46,XY,t(2;6)(p11.2;p25),t(9;?21)(p13;p11.2),del(11)(p12p15)[4] 46,XX,add(2)(q32),del(3)(p24p26),del(9)(q13q34),add(15)(q22),add(15) (q22),add(16)(q24),add(20)(q12)[2] 46,XX,add(2)(p22),del(10)(q21q26),add(22)(q13)[2] 46,XX,add(1)(p36.1),add(2)(p21),add(9)(p22)[1] 46,XX,add(1)(p36.1),add(3)(q21),del(6)(q22q25),del(8)(q21q24),add(12) (p12)[1] 46,XX,t(3;11)(?q22;?p11.2)[13] 46,XX,t(2;5)(p21;q35),inv(16)(p11.2q24)[10] 46,XX,add(7)(p22)[1] 46,XX,inv(14)(q21q23)[1] 46,XX,der(4)t(4;?),(q35;?)del(7)(p11.2p15)[18] 47,XX,del(X)(q13q28),der(1)t(1;?)(q12;?),þder(1)t(1;?)(q21;?),der(4)t(4;?) (q35;?),del(7)(p11.2p15)[1] 46,XY,t(2;12)(q24;p12)[9] 46,XY,der(9)t(9;?17)(q34;?q11),t(13;16)(q16;q24),14,i(14)(q10), 17,þ3mar[20] 46,XX,t(5;22)(q11;q13)[3] 46,XX,t(1;5)(q12;q33)[14] 46,XX,add(4)(p12),add(4)(p12),add(17)(p11.2)[1] 46,XX,add(4)(p12),del(4)(q21q35),13,add(17)(p11.2),þmar[2] 46,XY,t(2;3)(p16;p25)[20] 47,XX,þ21[13] 47,idem,?del(2)(p23p25),t(9;22)(q34;q11),add(17)(q24),þ21[5] 47,XX,19,þ21,þmar[1] 47,XX,der(1)t(1;?der(9)t(9;22)(q34;q11)(p21;q22)).del(3)(p21p26), add(19)(p13),þ21,der(22)t(9;22)(q34;q11)[1] 47,XY,þ2[19] 47,XY,1,þ2r[20] 47w48,XY,þ1w3mar[5]
Abbreviations: aCGH, array-based comparative genomic hybridization; NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; NS, not significant gain/loss by CGHcall; S, significant gain/loss by CGHcall [20]; PC, pericentromeric regions of uncertain significance due to limited BAC coverage; CNV-rich, copy number variationerich areas of uncertain significance due to lack of germ-line controls. a Each gain or loss is designated as S, statistically validated by Nexus and CGHcall (see Materials and Methods).
3.2. Cytogenetics Abnormal karyotypes at relapse in these 17 patients were complex (defined as three or more aberrations) in eight samples (47%); 9 patients had two or fewer abnormalities. Only one patient had a complex karyotype containing a deletion of chromosome 7. No patients had chromosome aberrations of 5q or 11q23 considered typical for secondary AML (Table 2).
3.3. Genomic studies To determine whether cytogenetic aberrations were present in NK AML diagnostic samples but were
undetected by conventional diagnostics, we performed high-resolution aCGH on paired NK AML diagnostic and AK AML relapse cells from the same patient. As shown in Figure 1, multiple cytogenetic aberrations were identified by aCGH in the AK AML relapse sample, which were not seen in the diagnostic NK AML sample. We then performed aCGH analyses of the remaining 16 diagnostic NK AML cells. Despite the identification of multiple potential aberrations in these samples, no significant copy number variations were deemed significant by additional band or statistical analysis. Although 21p11.2e11.1 and 9p13.3e11.2 losses were somewhat consistently found in diagnostic NK-AK AML samples, these chromosomal regions are pericentromeric with known poor BAC
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Fig. 1. Chromosomal abnormalities determined by array-CGH in the same patient at the time of diagnostic NK AML (top) and relapsed AK AML (bottom). The following aberrations were detected at the time of relapse: 1q31.3eq44 (gain), 16q12.2eq13 (loss), and 22q13.2e13.33 (loss). These aberrations were not identified in the diagnostic sample. NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia.
coverage, and therefore they more likely reflect the limitations of our platform rather than true copy number losses (Table 2).
of marker expression (specifically gains of one to three antigens), and three cases showed loss of one antigen at relapse but without definite evidence of new evolving populations/subpopulations (data not shown).
3.4. Immunophenotype We reviewed paired results of flow cytometry immunophenotyping at diagnosis and relapse, specifically looking for marker expression changes reflecting evolving of new blastic population/subpopulations or complete switching of the blastic immunophenotype [21]. Among 16 paired samples, a significant change in the overall AML immunophenotype was identified in only one case with complete switch to monocytic immunophenotype (myeloid to monocytic switch). Five other cases demonstrated some changes
3.5. Gene mutation analysis We also assessed NPM1 and FLT3 gene mutation results on NK-AK AML patient samples at diagnosis and relapse. NPM1 and/or FLT3 mutational status was available on 12 paired samples. Ten (83%) demonstrated the same NPM1 and/or FLT3 mutational status at relapse as in the original AML sample. Two paired patient samples did not. These two cases both had diagnostic NPM1-positive, FLT3-negative NK AML and relapsed NPM1-positive,
Table 3 NPM1 and FLT3 mutations at NK-AK AML diagnosis and relapse Patient no.
Diagnosis NK AML (NPM1/FLT3)
Relapse AK AML (NPM1/FLT3)
Same? (Y/N)
02-1407 04-0391 03-875 04-1272 06-130 05-1177 05-1517 04-0714 04-967 04-0071 03-106 05-210 06-614 06-394 03-1152 07-924 08-450
ND/ND Pos/Neg Pos/ITD 401e402 bp Pos/ITD 386e387 bp Pos/ITD 349e350 bp Neg/ITD 372e373 bp Pos/ITD 366e367 bp Pos/ITD 375e376 bp Neg/ITD 361e362 bp Pos/Neg Pos/Neg Pos/ITD 366e367 bp Pos/Neg Pos/Neg ND/ND Pos/Neg Pos/Neg
ND/ND ND/ND ND/ND ND/ITD Pos/ITD 349e350 bp Neg/ITD 372e373 bp Pos/ITD 366e367 bp ND/ITD ND/ITD Pos/TKD D835 ND/ND ND/ITD 366e367 bp Pos/Neg Neg/Neg ND/ND Pos/Neg Pos/ITD 353e353 bp
ND ND ND Y Y Y Y Y Y N ND Y Y Y ND Y N
Abbreviations: NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; Pos, mutation detected; Neg, mutation not detected; ND, not determined; NPM1, nucleophosmin-1 mutation; ITD, FLT3 internal tandem duplication; TKD, FLT3 tyrosine kinase domain mutation.
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Table 4 Comparison of clinical characteristics of NK AML patients with AK vs. NK AML relapse Characteristic
NK-AK (n 5 30)
NK-NK (n 5 30)
P value
Age (y), median (range/95% CI) Sex FAB classification
64.5 (21e81/58e70) 15 M, 15 F 1 M0, 10 M1, 11 M2, 6 M4, 1 M5a, 11 M2, 6 M4, 1 M5a
69.5 (37e83/66e74) 18 M, 12 F 9 M1, 11 M2, 7 M4, 2 M5a, 1 M5b
0.061 0.604 1.000
17.8 (0.57e138.1/10.1e50.5) 4.7 (0.25e192/4.1e12.9)
12.1 (0.83e249/6.3e27.8) 3.87 (1.13e97.6/2.8e7.4)
0.268 0.228
72 (22e95/59.5e83) 71 (3.5e97/40e81) 489 (325e574) 374 (250e457) 98.5 (58e176)
66 (22e93/52e78) 39 (4.5e91.5, 25e54) 497 (342e667) 266 (217e301) 205 (118e369)
0.412 0.017 0.640 0.119 0.002
WBC Diagnosis (109/L), median (range, 95% CI) Relapse (109/L), median (range, 95% CI) Blasts Diagnosis (%), median (range/95% CI) Relapse (%), median (range, 95% CI) Overall survival (d), median (95% CI) Time to relapse (d), median (95% CI) Event-free survival (d), median (95% CI)
Abbreviations: NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; 95% CI, 95% confidence interval; FAB, Frenche AmericaneBritish; WBC, white blood cell count. P values in bold font are considered statistically significant (P!0.05).
FLT3-mutated AK AML. One of these two patients (patient 08-450) was also noted via immunophenotyping to have a new relapsed AML population (Table 3; data not shown). 3.6. Overall clinical outcomes To confirm that our patients were representative of NKAK AML patients reported in other studies, we retrospectively examined the clinical outcomes of all 60 consecutive relapsed diagnostic NK AML patients treated at our institute between 1991 and 2009. Thirty had NK AML at relapse, and 30 had AK AML. As compared with NK-NK AML, NK-AK AML patients were younger (P 5 0.061) and had higher marrow blasts at relapse (71% vs. 39%, P 5 0.017). Overall survival and time to relapse did not differ; however, event-free survival was significantly shorter after AK AML (98.5 vs. 205 days, P 5 0.002) than after NK AML relapse, a finding consistent with prior reports (Table 4; Fig. 2).
4. Discussion We examined immunophenotype profiles and NPM1/ FLT3 gene mutation status in 17 paired NK-AK AML samples at diagnosis and relapse. In contrast with prior studies demonstrating a high frequency of immunophenotypic [21] and gene mutation changes (specifically NRAS, FLT3, and TP53) between AML diagnosis and relapse [22], we found that most paired NK-AK AML samples (O83%) did not demonstrate significant antigen or NPM1/FLT3 mutational changes over time. We did not, however, specifically evaluate NRAS or TP53 mutations in these samples. Only one patient (patient 08-450) out of 17 clearly had a different AML population by both surface antigen expression and FLT3 mutation status at relapse versus diagnosis. This suggests the possibility that in many cases, AK AML cells do not arise not from de novo secondary leukemogenesis but rather from clonal evolution of NK AML cells with acquisition of novel cytogenetic
Fig. 2. Clinical outcomes of NK AML patients with AK AML (n 5 30) versus NK AML (n 5 30) at relapse, specifically (A) overall survival, (B) time to relapse, and (C) event-free survival, defined as time between relapse and death from any cause, plotted as Kaplan-Meier curves. NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia.
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changes, as seen in chronic myeloid leukemia blast crisis and other leukemia types [23e25]. All 17 NK AML diagnostic samples were analyzed by means of a high-resolution 19K BAC aCGH specifically looking for cryptic cytogenetic aberrations below the resolution of conventional karyotypic analysis [26,27]. Prior investigators have demonstrated the ability of highresolution aCGH to detect cryptic chromosomal aberrations in primary NK AML samples [15,28,29]. This highresolution aCGH platform was selected on the basis of previous data demonstrating its accuracy in identifying cytogenetic abnormalities in the human HL60 AML cell line with fewer outliers and less technical noise than oligonucleotide-based CGH [28]. Excluding losses located in pericentromeric regions with poor BAC coverage, we identified no cytogenetic aberrations by aCGH at NK AML diagnosis that were missed by conventional cytogenetics. To confirm our methods, aCGH was performed both at diagnosis and relapse in one patient and identified clear aberrations only in AK AML cells. The fact that no recurrent aberrations were identified among all 17 NK AML diagnostic samples is consistent with prior reports [9e11]. These results support acquisition of novel genetic hits by preexisting NK AML cells as the most frequent etiology of subsequent AK AML relapse, rather than de novo leukemogenesis. However, we could not rule out the very rare presence of preexisting AK AML cells below the detection of aCGH. These data contrast with pediatric ALL, where small preexisting ALL subpopulations have been cited as the major relapse source. These latter studies, however, were performed in diagnosis and relapsed ALL samples of all karyotypes, not NK-AK adult AML, and may simply reflect differences between these very different leukemia diseases [8]. Whether these genetic hits arose as a result of selective pressure by and/or damage from interim chemotherapy [4,5,30] in rapidly proliferating AML cells remains to be determined [5]. Although overt therapy-related AML (t-AML) and myelodysplasia are very rare known complications of prior AML chemotherapy [31e36], at least one analysis has suggested that high-dose chemotherapy can frequently and rapidly induce t-AML-related genetic aberrations in many cells in a dose-dependent manner [37]. In vitro, etoposide exposure can lead to preferential accumulation of topoisomerase IIeDNA complexes in AML, resulting in damaged but viable leukemia cells [38]. Limitations of our study include small patient numbers, unavailability of all samples (dating back to 1991) for paired diagnosiserelapse and/or germ-line analyses, and the use of the high-resolution aCGH platform. As other whole genome approaches (i.e., oligonucleotidebased CGH or single nucleotide polymorphism arrays) or interphaseefluorescence in situ hybridization may not be significantly more sensitive than aCGH in detecting very rare AML populations, especially in the absence of germline samples [39], further confirmation of our results may best be performed by using sample-specific PCR primers
to identify individual mutations at time of AK AML relapse in prior NK AML samples (i.e., molecular backtracking experiments) [8]. Alternatively, transcriptional profiling (next-generation sequencing in NK AML cells) may also be helpful [40]. Similar to prior studies, relapse after NK AML diagnosis in our patients was associated with chemoresistance and poor subsequent survival [4,6,41]. Further studies to determine whether host factors (i.e., polymorphisms or defects in DNA repair genes) place some patients at higher risk for AK AML relapse after chemotherapy are warranted [42]. Additional characterization of the genetic aberrations underlying NK-AK AML may also reveal novel therapeutic targets [43]. Acknowledgments This research was supported in part by the Szefel Leukemia Research Fund (RPCI) and a National Cancer Institute Cancer Center support grant (CA016156). Thanks to Daniel P. Gaile, Lori Shepherd, Song Liu, Jeff Miecznikowski (SUNY-UB and RPCI Biostatistics), Paul Wallace (RPCI Flow Cytometry), AnneMarie W. Block (RPCI Cytogenetics), Maurice Barcos (RPCI Hematopathology), Marc S. Halfon (SUNY-UB COE), and the RPCI Leukemia Service for excellent patient care. References [1] Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998;92:2322e33. [2] Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100: 4325e36. [3] Breems DA, Van Putten WL, Huijgens PC, Ossenkoppele GJ, Verhoef GE, Verdonck LF, et al. Prognostic index for adult patients with acute myeloid leukemia in first relapse. J Clin Oncol 2005;23: 1969e78. [4] Kern W, Haferlach T, Schnittger S, Ludwig WD, Hiddemann W, Schoch C. Karyotype instability between diagnosis and relapse in 117 patients with acute myeloid leukemia: implications for resistance against therapy. Leukemia 2002;16:2084e91. [5] Testa JR, Mintz U, Rowley JD, Vardiman JW, Golomb HM. Evolution of karyotypes in acute nonlymphocytic leukemia. Cancer Res 1979;39:3619e27. [6] Estey E, Keating MJ, Pierce S, Stass S. Change in karyotype between diagnosis and first relapse in acute myelogenous leukemia. Leukemia 1995;9:972e6. [7] Garson OM, Hagemeijer A, Sakurai M, Reeves BR, Swansbury GJ, Williams GJ, et al. Cytogenetic studies of 103 patients with acute myelogenous leukemia in relapse. Cancer Genet Cytogenet 1989; 40:187e202. [8] Mullighan CG, Phillips LA, Su X, Ma J, Miller CB, Shurtleff SA, et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 2008;322:1377e80.
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[26] Michels E, De Preter K, Van Roy N, Speleman F. Detection of DNA copy number alterations in cancer by array comparative genomic hybridization. Genet Med 2007;9:574e84. [27] Casas S, Aventin A, Fuentes F, Vallespi T, Granada I, Carrio A, et al. Genetic diagnosis by comparative genomic hybridization in adult de novo acute myelocytic leukemia. Cancer Genet Cytogenet 2004;153: 16e25. [28] Nowak NJ, Miecznikowski J, Moore SR, Gaile D, Bobadilla D, Smith DD, et al. Challenges in array comparative genomic hybridization for the analysis of cancer samples. Genet Med 2007;9:585e95. [29] Cuneo A, Bigoni R, Cavazzini F, Bardi A, Roberti MG, Agostini P, et al. Incidence and significance of cryptic chromosome aberrations detected by fluorescence in situ hybridization in acute myeloid leukemia with normal karyotype. Leukemia 2002;16:1745e51. [30] Rowley JD, Potter D. Chromosomal banding patterns in acute nonlymphocytic leukemia. Blood 1976;47:705e21. [31] Ogasawara T, Yasuyama M, Kawauchi K. Therapy-related myelodysplastic syndrome with monosomy 5 after successful treatment of acute myeloid leukemia (M2). Am J Hematol 2005;79:136e41. [32] Ratain MJ, Rowley JD. Therapy-related acute myeloid leukemia secondary to inhibitors of topoisomerase II: from the bedside to the target genes. Ann Oncol 1992;3:107e11. [33] Krishnan A, Bhatia S, Slovak ML, Arber DA, Niland JC, Nademanee A, et al. Predictors of therapy-related leukemia and myelodysplasia following autologous transplantation for lymphoma: an assessment of risk factors. Blood 2000;95:1588e93. [34] Arana-Yi C, Block AW, Sait SN, Ford LA, Barcos M, Baer MR. Therapy-related myelodysplastic syndrome and acute myeloid leukemia following treatment of acute myeloid leukemia: possible role of cytarabine. Leuk Res 2008;32:1043e8. [35] Borthakur G, Estey AE. Therapy-related acute myelogenous leukemia and myelodysplastic syndrome. Curr Oncol Rep 2007;9:373e7. [36] Seiter K, Feldman EJ, Sreekantaiah C, Pozzuoli M, Weisberger J, Liu D, et al. Secondary acute myelogenous leukemia and myelodysplasia without abnormalities of chromosome 11q23 following treatment of acute leukemia with topoisomerase IIebased chemotherapy. Leukemia 2001;15:963e70. [37] Basecke J, Podleschny M, Becker A, Seiffert E, Schwiers I, Schwiers R, et al. Therapy-associated genetic aberrations in patients treated for non-Hodgkin lymphoma. Br J Haematol 2008;141:52e9. [38] Wang Y, Zhou R, Liliemark J, Gruber A, Lindemalm S, Albertioni F, et al. In vitro topo IIeDNA complex accumulation and cytotoxicity of etoposide in leukaemic cells from patients with acute myelogenous and chronic lymphocytic leukaemia. Leuk Res 2001;25:133e40. [39] Maciejewski JP, Mufti GJ. Whole genome scanning as a cytogenetic tool in hematologic malignancies. Blood 2008;112:965e74. [40] Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009;361:1058e66. [41] Kern W, Schoch C, Haferlach T, Braess J, Unterhalt M, Wormann B, et al. Multivariate analysis of prognostic factors in patients with refractory and relapsed acute myeloid leukemia undergoing sequential high-dose cytosine arabinoside and mitoxantrone (S-HAM) salvage therapy: relevance of cytogenetic abnormalities. Leukemia 2000;14:226e31. [42] Kuptsova N, Kopecky KJ, Godwin J, Anderson J, Hoque A, Willman CL, et al. Polymorphisms in DNA repair genes and therapeutic outcomes of AML patients from SWOG clinical trials. Blood 2007;109:3936e44. [43] Staal F, de Ridder D, Szczepanski T, Schonewille T, van der Linden E, van Wering E, et al. Genome-wide expression analysis of paired diagnosis-relapse samples in ALL indicates involvement of pathways related to DNA replication, cell cycle and DNA repair, independent of immune phenotype. Leukemia 2010;24:491e9.
Genomic, immunophenotypic, and NPM1/FLT3 mutational studies on 17 patients with normal karyotype acute myeloid leukemia (AML) followed by aberrant karyotype AML at relapse Eunice S. Wanga,*, Sheila N.J. Saitb, David Goldc,d, Terry Mashtarec,d, Petr Starostikb, Laurie Ann Forda, Meir Wetzlera, Norma J. Nowake, George Deebb a Leukemia Service, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263 c Department of Biostatistics, Roswell Park Cancer Institute, Buffalo, NY 14263 d Department of Biostatistics, SUNY-UB School of Public Health, Buffalo, NY 14214 e Department of Cancer Prevention, Roswell Park Cancer Institute, Buffalo, NY 14263 Received 23 February 2010; received in revised form 28 June 2010; accepted 2 July 2010
b
Abstract
Normal karyotype (NK) is the most common cytogenetic group in acute myeloid leukemia (AML) diagnosis; however, up to 50% of these patients at relapse will have aberrant karyotype (AK) AML. To determine the etiology of relapsed AK AML cells, we evaluated cytogenetic, immunophenotypic, and molecular results of 17 patients with diagnostic NK AML and relapsed AK AML at our institute. AK AML karyotype was diverse, involving no favorable and largely (8 of 17) complex cytogenetics. Despite clear cytogenetic differences, immunophenotype and NPM1/FLT3 gene mutation status did not change between presentation and relapse in 83% (10 of 12) and 94% (15 of 16) cases, respectively. High-resolution array-based comparative genomic hybridization (aCGH) performed via paired aCGH on NK AML and AK AML samples from the same patient confirmed cytogenetic aberrations only in the relapse sample. Analysis of 16 additional diagnostic NK AML samples revealed no evidence of submicroscopic aberrations undetected by conventional cytogenetics in any case. These results favor evolution of NK AML leukemia cells with acquisition of novel genetic changes as the most common etiology of AK AML relapse as opposed to secondary leukemogenesis. Additional studies are needed to confirm whether AK AML cells represent selection of rare preexisting clones below aCGH detection and to further characterize the molecular lesions found at time of AK AML relapse. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Acute myeloid leukemia (AML) is a rapidly proliferating neoplasm of immature hematopoietic cells. Diagnostic karyotype is well established as the best independent prognostic factor for AML for complete remission as well as for disease-free and overall survival. Normal karyotype (NK) AML is the most common AML cytogenetic group, comprising 40e49% of adult and 20e25% of pediatric AML diagnoses. NK AML is associated with an intermediate prognosis, defined as a 5-year survival rate of 24e40% [1,2]. Despite the importance of diagnostic karyotype [3], however, 25e51% of NK AML patients will have * Corresponding author. Tel.: (716) 845-3544; fax: (716) 845-8741. E-mail address: [email protected] (E.S. Wang). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.07.117
AML associated with aberrant karyotype (AK) at relapse [4e7]. It is unknown how these previously cytogenetically normal AML cells “acquire” additional genetic abnormalities [5]. One possibility is that AK AML cells were present but undetected at time of NK AML diagnosis as a result of low cell frequency and the relative insensitivity of conventional cytogenetic analyses. Recent analyses have shown that most relapsed pediatric acute lymphocytic leukemia (ALL) disease arises from minor tumor subpopulations present but undetected at time of diagnosis [8]. Alternatively, AK AML cells could have arisen from acquired genetic changes to preexisting NK AML cells. Finally, AK AML cells may represent secondary de novo leukemia cells. To elucidate the etiology of AK AML after NK AML, we identified 17 patients with diagnostic NK AML and
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relapsed AK AML (designated NK-AK AML) treated at our institute. We compared cytogenetic and immunophenotypic profiles, and NPM1/FLT3 gene mutation status from patient samples at diagnosis and relapse. In addition, we performed high-resolution array-based comparative genomic hybridization (aCGH) on all diagnostic NK AML samples specifically looking for chromosomal aberrations that might have been present but undetected by conventional karyotyping [9e11].
2. Materials and methods 2.1. Patient samples Under an IRB-approved protocol, we reviewed data on 301 consecutive NK AML patients diagnosed at our institute between 1991 and 2009. Clinical outcomes were defined per Cheson et al. [12]. Relapse was defined as the finding of O5% marrow blasts not attributable to another cause or to extramedullary disease. Event-free survival was defined as time between relapse and death from any cause. Sixty patients had karyotype information at both diagnosis and time of disease progression and/or relapse. Of these, 30 had NK AML at diagnosis and AK AML at relapse (NK-AK AML), and 30 had NK AML at both diagnosis and relapse (NK-NK AML). Seventeen NK-AK AML patients diagnosed between December 2002 and April 2008 were selected for further analysis on the basis of availability of stored cells at NK AML diagnosis containing 45% blast cells, which is considered the minimum unsorted cell threshold to undergo aCGH analysis. Because all 17 NK AML cases lacked recurrent genetic abnormalities recognized by World Health Organization classification, they were classified morphologically according to FrencheAmericaneBritish classification. Multiparameter four-color flow cytometry [13] was reviewed on all available diagnostic and relapsed AML samples. Genomic DNA from fresh or cryopreserved NK AML marrow aspirate cells was also analyzed for FLT3 and NPM1 mutations. FLT3 gene internal tandem duplications and tyrosine kinase domain mutations were performed by polymerase chain reaction (PCR) methods as previously described [14]. NPM1 mutational analysis was performed by PCR amplification of insertions occurring at the 50 intron/exon boundary followed by separation/sizing by capillary electrophoresis on an ABI 3130xl Genetic Analyzer. Both forward and reverse PCR primers were hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxyfluorescein (FAM) labeled, respectively. A wild-type result consisted of HEX- and FAM-labeled PCR products at 122 bp and 125 bp, respectively. NPM1 mutants yielded FAM- and HEX-labeled PCR products that were greater in size than wild type. Sequences were as follows: NPM1 F fam2 5 50 -6-FAM-TTT-CTT-TTT-TTT-TTT-TTC-CAGGC -30 ; NPM1R3 hex 5 50 -HEX-AGA-TAT-CAA-CTGTTA-CAG-AAA-TGA-AAT-AAG-30 .
2.2. Array-based comparative genomic hybridization All patient samples were procured after informed patient consent was obtained under an institutional review board (IRB) protocol. aCGH was performed under a separate approved IRB protocol. Genomic DNA was prepared using FlexiGene DNA Isolation kit (Qiagen, Valencia, CA) from O1e2 106 unsorted AML patient marrow mononuclear cells. aCGH utilizing 19K bacterial artificial chromosomes (RP11 bacterial artificial chromosome [BAC] library) with image analysis was performed [15e17]. Genomic aberrations were identified by Nexus software, version 3.0 (BioDiscovery, El Segundo, CA), with a built-in Rank Segmentation algorithm and a cutoff of þ0.15/0.15 based on prior studies of human leukemia samples [18,19]. All cases showed chromosome X and Y gain/loss signals compatible with the patient’s sex based on this cutoff and were compared with sex-matched normal controls. Statistical significance of copy gains/ deletions identified by Nexus was inferred with CGHcall algorithm, using an 80% threshold on the conditional posterior probability in favor of gain or deletion, respectively [20].
3. Results 3.1. Clinical characteristics Characteristics of the 17 NK-AK AML patients evaluated here are summarized in Table 1. All patients received induction therapy with cytarabine- and anthracycline-based induction AML regimens before relapse or recurrence. Overall time and time to AK AML development were 470 213 days and 294 198 days, respectively. Eventfree survival (or survival after AK AML diagnosis) was only 96 64 days. Table 1 NK-AK AML patient characteristicsa Characteristic
Value
Age (y), range (median) Sex (M/F) FAB classification WBC (109/L) Diagnosis Relapse Blasts (%) Diagnosis Relapse Overall survival (d) Time to AK AML (d) Event-free survival (d)
68 (21e81) 9/8 7 M1, 7M2, 2 M4, 1 M5a 37.7 39 (2.01e138.1) 4.76 53 (0.25e192.1) 80 16 (48e95) 73.5 23 (23e97) 470 213 (240e1,110) 294 198 (105e986) 96 64 (18e202)
Abbreviations: NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; FAB, FrencheAmericaneBritish; WBC, white blood cell count. a Data are expressed as median standard deviation (range) unless indicated otherwise.
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Table 2 Cytogenetic abnormalities detected by aCGH and karyotype in NK-AK AML samplesa Patient no.
Diagnosis karyotype
Diagnosis aCGH analysis (band/gain or loss)
02-1407 04-0391
46,XX[20] 46,XY[13]
5q13.2 gain (NS, CNV rich) 21p11.1 loss (NS)
03-0875
46,XY[20]
04-1272 06-0130
46,XY[20] 46,XX[20]
11q22.3 gain (NS) 16p11.2e11.1 gain (NS) 21p11.1 loss (NS) No gains/losses 21 p11.2e11.1 gain (NS) 9p13.3e11.2 loss (S, PC)
05-1177 05-1517
46,XX[25] 46,XX[20]
1q31.1e32.1 (NS) No gains/losses
04-0714
46,XX[21]
1q21.1e21.2 loss (NS)
04-0967 04-0071
46,XY[19] 46,XY[16]
9p13.3e11.2 loss (S, PC) No gains/losses
03-0106 05-0210
46,XX[25] 46,XX[20]
21p11.2e11.1 gain (NS, PC) 21p11.2e11.1 gain (NS) (PC)
06-0614 06-0394
46,XY[20] 46,XX[20]
No gains/losses No gains/losses
03-1152 07-0924
46,XY[20] 46,XY[20]
08-0450
46,XY[20]
9p13.1e11.2 loss (S, PC) 1q12e21.1 gain (NS, PC, CNV-rich) 21p11.2e11.1 gain (NS) 7p21.1 loss (NS)
Relapse karyotype 46,XX,del(11)(p11.2p14)[19] 46,XY,t(11;16)(p13;q22)[5] 46,XY,t(9;11)(p13;q13),t(12;22)(q13;q13)[9] 46,XY,t(13;17)(q14;q11.2)[14]
46,XY,t(2;3)(p21;p21),del(3)(q21q26),t(7;11)(p21;p14)[19] 46,XY,t(2;6)(p11.2;p25),t(9;?21)(p13;p11.2),del(11)(p12p15)[4] 46,XX,add(2)(q32),del(3)(p24p26),del(9)(q13q34),add(15)(q22),add(15) (q22),add(16)(q24),add(20)(q12)[2] 46,XX,add(2)(p22),del(10)(q21q26),add(22)(q13)[2] 46,XX,add(1)(p36.1),add(2)(p21),add(9)(p22)[1] 46,XX,add(1)(p36.1),add(3)(q21),del(6)(q22q25),del(8)(q21q24),add(12) (p12)[1] 46,XX,t(3;11)(?q22;?p11.2)[13] 46,XX,t(2;5)(p21;q35),inv(16)(p11.2q24)[10] 46,XX,add(7)(p22)[1] 46,XX,inv(14)(q21q23)[1] 46,XX,der(4)t(4;?),(q35;?)del(7)(p11.2p15)[18] 47,XX,del(X)(q13q28),der(1)t(1;?)(q12;?),þder(1)t(1;?)(q21;?),der(4)t(4;?) (q35;?),del(7)(p11.2p15)[1] 46,XY,t(2;12)(q24;p12)[9] 46,XY,der(9)t(9;?17)(q34;?q11),t(13;16)(q16;q24),14,i(14)(q10), 17,þ3mar[20] 46,XX,t(5;22)(q11;q13)[3] 46,XX,t(1;5)(q12;q33)[14] 46,XX,add(4)(p12),add(4)(p12),add(17)(p11.2)[1] 46,XX,add(4)(p12),del(4)(q21q35),13,add(17)(p11.2),þmar[2] 46,XY,t(2;3)(p16;p25)[20] 47,XX,þ21[13] 47,idem,?del(2)(p23p25),t(9;22)(q34;q11),add(17)(q24),þ21[5] 47,XX,19,þ21,þmar[1] 47,XX,der(1)t(1;?der(9)t(9;22)(q34;q11)(p21;q22)).del(3)(p21p26), add(19)(p13),þ21,der(22)t(9;22)(q34;q11)[1] 47,XY,þ2[19] 47,XY,1,þ2r[20] 47w48,XY,þ1w3mar[5]
Abbreviations: aCGH, array-based comparative genomic hybridization; NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; NS, not significant gain/loss by CGHcall; S, significant gain/loss by CGHcall [20]; PC, pericentromeric regions of uncertain significance due to limited BAC coverage; CNV-rich, copy number variationerich areas of uncertain significance due to lack of germ-line controls. a Each gain or loss is designated as S, statistically validated by Nexus and CGHcall (see Materials and Methods).
3.2. Cytogenetics Abnormal karyotypes at relapse in these 17 patients were complex (defined as three or more aberrations) in eight samples (47%); 9 patients had two or fewer abnormalities. Only one patient had a complex karyotype containing a deletion of chromosome 7. No patients had chromosome aberrations of 5q or 11q23 considered typical for secondary AML (Table 2).
3.3. Genomic studies To determine whether cytogenetic aberrations were present in NK AML diagnostic samples but were
undetected by conventional diagnostics, we performed high-resolution aCGH on paired NK AML diagnostic and AK AML relapse cells from the same patient. As shown in Figure 1, multiple cytogenetic aberrations were identified by aCGH in the AK AML relapse sample, which were not seen in the diagnostic NK AML sample. We then performed aCGH analyses of the remaining 16 diagnostic NK AML cells. Despite the identification of multiple potential aberrations in these samples, no significant copy number variations were deemed significant by additional band or statistical analysis. Although 21p11.2e11.1 and 9p13.3e11.2 losses were somewhat consistently found in diagnostic NK-AK AML samples, these chromosomal regions are pericentromeric with known poor BAC
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Fig. 1. Chromosomal abnormalities determined by array-CGH in the same patient at the time of diagnostic NK AML (top) and relapsed AK AML (bottom). The following aberrations were detected at the time of relapse: 1q31.3eq44 (gain), 16q12.2eq13 (loss), and 22q13.2e13.33 (loss). These aberrations were not identified in the diagnostic sample. NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia.
coverage, and therefore they more likely reflect the limitations of our platform rather than true copy number losses (Table 2).
of marker expression (specifically gains of one to three antigens), and three cases showed loss of one antigen at relapse but without definite evidence of new evolving populations/subpopulations (data not shown).
3.4. Immunophenotype We reviewed paired results of flow cytometry immunophenotyping at diagnosis and relapse, specifically looking for marker expression changes reflecting evolving of new blastic population/subpopulations or complete switching of the blastic immunophenotype [21]. Among 16 paired samples, a significant change in the overall AML immunophenotype was identified in only one case with complete switch to monocytic immunophenotype (myeloid to monocytic switch). Five other cases demonstrated some changes
3.5. Gene mutation analysis We also assessed NPM1 and FLT3 gene mutation results on NK-AK AML patient samples at diagnosis and relapse. NPM1 and/or FLT3 mutational status was available on 12 paired samples. Ten (83%) demonstrated the same NPM1 and/or FLT3 mutational status at relapse as in the original AML sample. Two paired patient samples did not. These two cases both had diagnostic NPM1-positive, FLT3-negative NK AML and relapsed NPM1-positive,
Table 3 NPM1 and FLT3 mutations at NK-AK AML diagnosis and relapse Patient no.
Diagnosis NK AML (NPM1/FLT3)
Relapse AK AML (NPM1/FLT3)
Same? (Y/N)
02-1407 04-0391 03-875 04-1272 06-130 05-1177 05-1517 04-0714 04-967 04-0071 03-106 05-210 06-614 06-394 03-1152 07-924 08-450
ND/ND Pos/Neg Pos/ITD 401e402 bp Pos/ITD 386e387 bp Pos/ITD 349e350 bp Neg/ITD 372e373 bp Pos/ITD 366e367 bp Pos/ITD 375e376 bp Neg/ITD 361e362 bp Pos/Neg Pos/Neg Pos/ITD 366e367 bp Pos/Neg Pos/Neg ND/ND Pos/Neg Pos/Neg
ND/ND ND/ND ND/ND ND/ITD Pos/ITD 349e350 bp Neg/ITD 372e373 bp Pos/ITD 366e367 bp ND/ITD ND/ITD Pos/TKD D835 ND/ND ND/ITD 366e367 bp Pos/Neg Neg/Neg ND/ND Pos/Neg Pos/ITD 353e353 bp
ND ND ND Y Y Y Y Y Y N ND Y Y Y ND Y N
Abbreviations: NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; Pos, mutation detected; Neg, mutation not detected; ND, not determined; NPM1, nucleophosmin-1 mutation; ITD, FLT3 internal tandem duplication; TKD, FLT3 tyrosine kinase domain mutation.
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Table 4 Comparison of clinical characteristics of NK AML patients with AK vs. NK AML relapse Characteristic
NK-AK (n 5 30)
NK-NK (n 5 30)
P value
Age (y), median (range/95% CI) Sex FAB classification
64.5 (21e81/58e70) 15 M, 15 F 1 M0, 10 M1, 11 M2, 6 M4, 1 M5a, 11 M2, 6 M4, 1 M5a
69.5 (37e83/66e74) 18 M, 12 F 9 M1, 11 M2, 7 M4, 2 M5a, 1 M5b
0.061 0.604 1.000
17.8 (0.57e138.1/10.1e50.5) 4.7 (0.25e192/4.1e12.9)
12.1 (0.83e249/6.3e27.8) 3.87 (1.13e97.6/2.8e7.4)
0.268 0.228
72 (22e95/59.5e83) 71 (3.5e97/40e81) 489 (325e574) 374 (250e457) 98.5 (58e176)
66 (22e93/52e78) 39 (4.5e91.5, 25e54) 497 (342e667) 266 (217e301) 205 (118e369)
0.412 0.017 0.640 0.119 0.002
WBC Diagnosis (109/L), median (range, 95% CI) Relapse (109/L), median (range, 95% CI) Blasts Diagnosis (%), median (range/95% CI) Relapse (%), median (range, 95% CI) Overall survival (d), median (95% CI) Time to relapse (d), median (95% CI) Event-free survival (d), median (95% CI)
Abbreviations: NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia; 95% CI, 95% confidence interval; FAB, Frenche AmericaneBritish; WBC, white blood cell count. P values in bold font are considered statistically significant (P!0.05).
FLT3-mutated AK AML. One of these two patients (patient 08-450) was also noted via immunophenotyping to have a new relapsed AML population (Table 3; data not shown). 3.6. Overall clinical outcomes To confirm that our patients were representative of NKAK AML patients reported in other studies, we retrospectively examined the clinical outcomes of all 60 consecutive relapsed diagnostic NK AML patients treated at our institute between 1991 and 2009. Thirty had NK AML at relapse, and 30 had AK AML. As compared with NK-NK AML, NK-AK AML patients were younger (P 5 0.061) and had higher marrow blasts at relapse (71% vs. 39%, P 5 0.017). Overall survival and time to relapse did not differ; however, event-free survival was significantly shorter after AK AML (98.5 vs. 205 days, P 5 0.002) than after NK AML relapse, a finding consistent with prior reports (Table 4; Fig. 2).
4. Discussion We examined immunophenotype profiles and NPM1/ FLT3 gene mutation status in 17 paired NK-AK AML samples at diagnosis and relapse. In contrast with prior studies demonstrating a high frequency of immunophenotypic [21] and gene mutation changes (specifically NRAS, FLT3, and TP53) between AML diagnosis and relapse [22], we found that most paired NK-AK AML samples (O83%) did not demonstrate significant antigen or NPM1/FLT3 mutational changes over time. We did not, however, specifically evaluate NRAS or TP53 mutations in these samples. Only one patient (patient 08-450) out of 17 clearly had a different AML population by both surface antigen expression and FLT3 mutation status at relapse versus diagnosis. This suggests the possibility that in many cases, AK AML cells do not arise not from de novo secondary leukemogenesis but rather from clonal evolution of NK AML cells with acquisition of novel cytogenetic
Fig. 2. Clinical outcomes of NK AML patients with AK AML (n 5 30) versus NK AML (n 5 30) at relapse, specifically (A) overall survival, (B) time to relapse, and (C) event-free survival, defined as time between relapse and death from any cause, plotted as Kaplan-Meier curves. NK, normal karyotype; AK, aberrant karyotype; AML, acute myeloid leukemia.
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changes, as seen in chronic myeloid leukemia blast crisis and other leukemia types [23e25]. All 17 NK AML diagnostic samples were analyzed by means of a high-resolution 19K BAC aCGH specifically looking for cryptic cytogenetic aberrations below the resolution of conventional karyotypic analysis [26,27]. Prior investigators have demonstrated the ability of highresolution aCGH to detect cryptic chromosomal aberrations in primary NK AML samples [15,28,29]. This highresolution aCGH platform was selected on the basis of previous data demonstrating its accuracy in identifying cytogenetic abnormalities in the human HL60 AML cell line with fewer outliers and less technical noise than oligonucleotide-based CGH [28]. Excluding losses located in pericentromeric regions with poor BAC coverage, we identified no cytogenetic aberrations by aCGH at NK AML diagnosis that were missed by conventional cytogenetics. To confirm our methods, aCGH was performed both at diagnosis and relapse in one patient and identified clear aberrations only in AK AML cells. The fact that no recurrent aberrations were identified among all 17 NK AML diagnostic samples is consistent with prior reports [9e11]. These results support acquisition of novel genetic hits by preexisting NK AML cells as the most frequent etiology of subsequent AK AML relapse, rather than de novo leukemogenesis. However, we could not rule out the very rare presence of preexisting AK AML cells below the detection of aCGH. These data contrast with pediatric ALL, where small preexisting ALL subpopulations have been cited as the major relapse source. These latter studies, however, were performed in diagnosis and relapsed ALL samples of all karyotypes, not NK-AK adult AML, and may simply reflect differences between these very different leukemia diseases [8]. Whether these genetic hits arose as a result of selective pressure by and/or damage from interim chemotherapy [4,5,30] in rapidly proliferating AML cells remains to be determined [5]. Although overt therapy-related AML (t-AML) and myelodysplasia are very rare known complications of prior AML chemotherapy [31e36], at least one analysis has suggested that high-dose chemotherapy can frequently and rapidly induce t-AML-related genetic aberrations in many cells in a dose-dependent manner [37]. In vitro, etoposide exposure can lead to preferential accumulation of topoisomerase IIeDNA complexes in AML, resulting in damaged but viable leukemia cells [38]. Limitations of our study include small patient numbers, unavailability of all samples (dating back to 1991) for paired diagnosiserelapse and/or germ-line analyses, and the use of the high-resolution aCGH platform. As other whole genome approaches (i.e., oligonucleotidebased CGH or single nucleotide polymorphism arrays) or interphaseefluorescence in situ hybridization may not be significantly more sensitive than aCGH in detecting very rare AML populations, especially in the absence of germline samples [39], further confirmation of our results may best be performed by using sample-specific PCR primers
to identify individual mutations at time of AK AML relapse in prior NK AML samples (i.e., molecular backtracking experiments) [8]. Alternatively, transcriptional profiling (next-generation sequencing in NK AML cells) may also be helpful [40]. Similar to prior studies, relapse after NK AML diagnosis in our patients was associated with chemoresistance and poor subsequent survival [4,6,41]. Further studies to determine whether host factors (i.e., polymorphisms or defects in DNA repair genes) place some patients at higher risk for AK AML relapse after chemotherapy are warranted [42]. Additional characterization of the genetic aberrations underlying NK-AK AML may also reveal novel therapeutic targets [43]. Acknowledgments This research was supported in part by the Szefel Leukemia Research Fund (RPCI) and a National Cancer Institute Cancer Center support grant (CA016156). Thanks to Daniel P. Gaile, Lori Shepherd, Song Liu, Jeff Miecznikowski (SUNY-UB and RPCI Biostatistics), Paul Wallace (RPCI Flow Cytometry), AnneMarie W. Block (RPCI Cytogenetics), Maurice Barcos (RPCI Hematopathology), Marc S. Halfon (SUNY-UB COE), and the RPCI Leukemia Service for excellent patient care. References [1] Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998;92:2322e33. [2] Byrd JC, Mrozek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood 2002;100: 4325e36. [3] Breems DA, Van Putten WL, Huijgens PC, Ossenkoppele GJ, Verhoef GE, Verdonck LF, et al. Prognostic index for adult patients with acute myeloid leukemia in first relapse. J Clin Oncol 2005;23: 1969e78. [4] Kern W, Haferlach T, Schnittger S, Ludwig WD, Hiddemann W, Schoch C. Karyotype instability between diagnosis and relapse in 117 patients with acute myeloid leukemia: implications for resistance against therapy. Leukemia 2002;16:2084e91. [5] Testa JR, Mintz U, Rowley JD, Vardiman JW, Golomb HM. Evolution of karyotypes in acute nonlymphocytic leukemia. Cancer Res 1979;39:3619e27. [6] Estey E, Keating MJ, Pierce S, Stass S. Change in karyotype between diagnosis and first relapse in acute myelogenous leukemia. Leukemia 1995;9:972e6. [7] Garson OM, Hagemeijer A, Sakurai M, Reeves BR, Swansbury GJ, Williams GJ, et al. Cytogenetic studies of 103 patients with acute myelogenous leukemia in relapse. Cancer Genet Cytogenet 1989; 40:187e202. [8] Mullighan CG, Phillips LA, Su X, Ma J, Miller CB, Shurtleff SA, et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 2008;322:1377e80.
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