Letters to the Editor
2058 significance in the younger cohort (57% for cases GNNK vs 19% for cases GNNK plus GNNK þ , P ¼ 0.04). The statistical significance was lost when PFS was considered, notwithstanding a persistent trend to significance (58% for cases with GNNK vs 25% for cases showing both GNNK plus GNNK þ , P ¼ 0.12). However, the absence of GNNK þ did highly condition both OS and PFS in the high cytogenetic risk subgroup (3-year OS 100% for cases GNNK vs 6% for those carrying both isoforms, P ¼ 0.01; PFS 66 vs 8%, P ¼ 0.05). Finally, all data about c-Kit (both ratio and GNNK þ absence) were analyzed in comparison with FLT3 and NPM1 mutations. In the whole series, 24% of cases showed an FLT3 mutation (65% FLT3-ITD and 35% D835); both the GNNK/ þ ratio and the GNNK þ absence did not correlate with the FLT3 mutational status. Four cases were NPM1-mutated and nine wild type; notwithstanding the small number of tested cases, in our series the c-Kit status did not correlate with NPM1 mutations. In conclusion, in this study, we demonstrated that a PCR assay detecting the two c-Kit GNNK isoforms represents a cheap, feasible and rapid molecular test able to identify AML cases with a worse prognosis. Our results show that this finding is independent of other already known prognostic factors, such as advanced age, high cytogenetic risk, chemoresistance, as shown also by the multivariate analysis (advanced age: P ¼ 0.41, high cytogenetic risk P ¼ 0.06, not CR P ¼ 0.0001, GNNK þ detection P ¼ 0.001). The oncogenic potential of the two c-Kit isoforms is still a matter of debate: Caruana et al.2 did not observe difference in ligand-binding affinity between the two isoforms. On the other hand, other authors demonstrated a more rapid internalization and a ubiquitin-mediated degradation of the GNNK form.6 If this observation implies a more rapid switch-off of the c-Kit activity, this phenomenon could be in accordance with our observations. The role of c-Kit in AML is particularly appealing for therapeutic options, considering the availability of several c-Kit inhibitors, such as imatinib and sorafenib. Low-dose Ara-C plus imatinib did not appear to be inferior in older AML patients in comparison with historic controls receiving myelosuppressive therapy, with median OS of 138 days and 20% of patients alive after 600 days.7 On the other hand, sorafenib (BAY 43-9006, Nexavar, Bayer, Germany) is a multikinase inhibitor with activity against Raf kinase and several receptor tyrosine kinases, including vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor, FLT3, Ret and c-Kit, the D816V mutants
excluded. Pronounced apoptosis was recently observed in blasts from patients with acute myeloid leukaemia.8 On the basis of the above reported data, it would be useful to screen all AML patients for GNNK and GNNK þ expression, in order to design ab initio a more aggressive therapeutic strategy, perhaps including transplantation options, for cases carrying both GNNK and GNNK þ c-Kit isoforms.
F Guerrini1, S Galimberti1, E Ciabatti1, S Brizzi1, R Testi1, A Pollastrini1, B Falini2 and M Petrini1 1 Department of Oncology, Transplant and Advances in Medicine, Section of Haematology, University of Pisa, Pisa, Italy and 2 Institute of Haematology, University of Perugia, Perugia, Italy E-mail:
[email protected] References 1 Cairoli R, Beghini A, Grillo G, Nadali G, Elice F, Ripamonti CB et al. Prognostic impact of c-KIT mutations in core binding factor leukaemias: an Italian retrospective study. Blood 2006; 107: 3463–3468. 2 Caruana G, Cambareri AC, Ashman LK. Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts. Oncogene 1999; 18: 5573–5581. 3 Galimberti S, Rossi A, Palumbo GA, Morabito F, Guerrini F, Vincelli I et al. FLT3 mutations do not influence MDR-1 gene expression in acute myeloid leukemia. Anticancer Res 2003; 23: 3419–3426. 4 Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005; 352: 254–266. Erratum in: N Engl J Med 2005; 352: 740. 5 Theou N, Tabone S, Saffroy R, Le Cesne A, Julie C, Cortez A et al. High expression of both mutant and wild-type alleles of c-kit in gastrointestinal stromal tumors. Biochim Biophys Acta 2004; 1688: 250–256. 6 Voytyuk O, Lennartsson J, Mogi A, Caruana G, Courtneidge S, Ashman LK et al. Src family kinases are involved in the differential signaling from two splice forms of c-Kit. J Biol Chem 2003; 278: 9159–9166. 7 Heidel F, Cortes J, Rucker FG, Aulitzky W, Letvak L, Kindler T et al. Results of a multicenter phase II trial for older patients with c-Kitpositive acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) using low-dose Ara-C and Imatinib. Cancer 2007; 109: 907–914. 8 Rahmani M, Davis EM, Bauer C, Dent P, Grant S. Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves downregulation of Mcl-1 through inhibition of translation. J Biol Chem 2005; 280: 35217–35227.
Single nucleotide polymorphism arrays complement metaphase cytogenetics in detection of new chromosomal lesions in MDS Leukemia (2007) 21, 2058–2061; doi:10.1038/sj.leu.2404745; published online 24 May 2007
In myelodysplastic syndromes (MDS) chromosomal aberrations have many clinical implications1,2 but a significant proportion of patients do not exhibit cytogenetic abnormalities, while in some patients, cytogenetic analysis may not provide an informative result. Recently, we investigated various possibilities to improve cytogenetic diagnosis of MDS. Currently, in MDS Leukemia
using routine metaphase cytogenetics (MC) defects are found only in 40–60% of patients,3 and MC analysis often yields uninformative results. We will share some of the important preliminary results here. While exploring high-density single nucleotide polymorphism DNA arrays (SNP-A) as genotyping tool, we realized that this technique can also be applied for detection of copy number (CN) changes, CN neutral loss of heterozygosity (LOH), also known as uniparental disomy (UPD), and allow for increased levels of genomic resolution. The resulting DNA-based SNP
Letters to the Editor
2059 ‘karyograms’ facilitate detection of chromosome defects without the need for dividing cells as in conventional cytogenetics. We hypothesized that chromosomal changes may be present in most MDS patients, and that such a cytogenetic technique with a higher resolution will result in detection of karyotypic defects in a higher proportion of patients. We have tested 66 patients with MDS (age range 26–86) and 29 healthy individuals (age range 27–61) used as controls (Table 1). The Affymetrix Gene Chip Mapping 50K Assay Kit (Affymetrix, Santa Clara, CA, USA) was used. We confirmed newly detected lesions using microsatellite (MS) analysis and determination of gene CN using a Real-Time TaqMan chemistry protocol. By analysis of heterozygous SNP calls within the X chromosome we estimated the accuracy of the array to be 499%. Expected differences in the signal intensity of regions affected by CN polymorphisms in controls were detected, and consequently, if found in MDS patients were not deemed significant. In general, 50K SNP-A confirmed the lesions identified by MC but also detected new, previously cryptic defects, including UPD (Table 2 and Figure 1). For example, SNP array analysis confirmed the presence of 2p and 5q deletion as seen in a patient with a 46,XY,del(2)(p11.2p15),del(5)(q13q33) karyotype (Figure 1a) and the presence of an extra copy of chromosome 13 in a patient with trisomy 13 (Figure 1b). When LOH was investigated by SNP-array, more subtle DNA variants could be identified, including microduplications and microdeletions (examples shown in Table 2). An unexpected finding was the identification of cytogenetically cryptic aberrations, including CN-neutral LOH (for example, 6p, Figure 1c). This type of LOH found by SNP-A was present despite having two copies of each respective chromosome. Importantly, this segmental LOH remains undetectable by traditional cytogenetic banding techniques, as it does not alter the chromosome banding pattern. SNP-A assessment of DNA CN was confirmed with TaqMan PCR-based quantitation using DNA from both marrow and lymphocytes (data not shown). The distinction between LOH resulting from deletions and that due to acquired UPD is apparent when comparing the SNP analysis for patients with deletions and UPD. The segmental LOH in UPD occurs in the presence of a diploid chromosome CN shown both by SNP array and MC (see, for example, UPD6p in Figure 1c) whereas, the ‘traditional’ LOH occurring in deletions is associated with loss of DNA CN in marrow cells.
Table 1
Identification of a high frequency of segmental LOH due to UPD is a new finding that has significant implications for the pathogenesis of MDS and it extends the previous observations of a high frequency of LOH in chromosome 1 using MS scanning.4 This mechanism of LOH has been described in many solid tumors, but has been rarely observed in myeloid malignancies.5 Importantly, LOH can also occur as a result of mitotic recombination during which segments of homologous chromosomes are exchanged, leading to UPD,6 a feature which is not recognizable in MC because the chromosome banding pattern remains preserved. While UPD is known as a mechanism leading to LOH affecting whole chromosomes in congenital abnormalities such as Prader–Willi and Angelman syndromes, formal proof for acquired segmental LOH in cancer has been described only in a limited number of reports.7,8 Deletions of chromosomal material could represent ‘unrepaired’ lesions while defects ‘repaired’ by mitotic recombination using the homologous chromosome would generate UPD and those lesions corrected using a nonhomologous chromosome could lead to translocations. We have shown that the presence of acquired segmental UPD in MDS is due to a somatic event; these results were independently confirmed by MS genotyping and PCR quantitation of locus CN. Fewer patients had normal karyotypes by SNP-A as compared to MC (12 vs 39%; Po0.001). In 69% of patients with normal MC, new chromosomal lesions were identified. In 80% of patients in whom MC was unsuccessful, SNP array analysis identified abnormalities. Moreover, additional lesions were found in 83% of patients with previously known defects (Table 1). Using morphologic diagnosis as the gold standard, the resulting sensitivity for detection of chromosomal changes in MDS by SNP-A was 83 vs 53% for cytogenetics. Moreover, SNP-A showed a higher proportion of patients with multiple chromosomal defects for SNP arrays and MC, respectively. Based on the hypothetical assumption that chromosomal aberrations detected by SNP-A carry a similar prognostic value to those detected by MC, array-based methods could result in a higher proportion of complex karyotypes (44 vs 6%; Po0.001) and consequently a higher IPSS score. Surprisingly, we found segmental LOH due to acquired UPD in 33% MDS patients by SNP array analysis. LOH was distributed across all WHO subtypes and was present both as a solitary lesion (9%), as well in association with additional defects (24%). Acquired UPD was present in regions frequently
Results of routine metaphase cytogenetics as compared to SNP-A based karyotyping
WHO subtypes (no. of patients)
RA (N ¼ 9) RCMD (N ¼ 11) MDS with isolated 5q-(N ¼ 1) RARS (N ¼ 5) RCMD-RS (N ¼ 8) RARS-t (N ¼ 4) RAEB-1 (N ¼ 7) RAEB-2 (N ¼ 10) AML (N ¼ 4) CMML-1 (N ¼ 4) CMML-2 (N ¼ 3) Controls (N ¼ 29)
Metaphase karyotyping
SNP karyotyping
Normal
Abnormal
NG
Normal
Abnormal
NI
38%
52%
10%
5%
90%
5%
41%
53%
6%
24%
76%
0%
33%
57%
10%
10%
80%
10%
57%
43%
0%
14%
86%
0%
N/A
N/A
N/A
87%
13%
0%
Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplastic syndromes; NI, not informative; NG, no growth; SNP-A, single nucleotide polymorphism DNA arrays. Leukemia
Letters to the Editor
2060 Table 2 Patient no.
Examples of previously cryptic lesions found in patients by SNP-A karyotyping Dx
MC result
Additional lesions found Gain
3 5 12
RA RA RCMD
Normal 47,XY,Y[20] Normal
15
RCMD
No growth
20 23 25
RCMD RARS RARS
48,XY,+8,+8[6]/46,XY[14] 46,XY,del(7)(q22q34)[4] No growth
66
CMML-2
45,XX,7[20]
Loss
UPD 6p21.1–pter 7q31.31–q31.33 13q31.1–q31.2
13q21.32 18q12.2 20p12.1 4p15.2 1p31.1 2p22.3
6q12–q13 20q11.22–q13.2 14q11.2
1q25.2–q25.3 14q24.2–qter 10p11.21–q11.22 17q11.2–qter
Abbreviations: MC, metaphase cytogenetics; SNP-A, single nucleotide polymorphism DNA arrays; UPD, uniparental disomy. Patients shown represent examples of individual results.
Figure 1 Detection and confirmation of SNP array results in patients with MDS. (a) An example of a whole genome data output. Each dot on the histogram represents the log 2 intensity ratio for each SNP locus. Each chromosome is coded with different color. The ratios are mapped horizontally in order of respective chromosomal location, corresponding to the short arm of chromosome 1 (far left) through the long arm of chromosome X (far right); Y chromosome DNA is not represented on the SNP array chip. The histogram shows three areas of DNA copy reduction corresponding to chromosomes 2, 5 and X respectively, indicating that only one copy of each chromosome is present. The SNP array findings are concordant with metaphase cytogenetic analysis that showed a 46,XY,del(2)(p11.2p15),del(5)(q13q33) karyotype. (b) MC-detected chromosomal abnormalities were confirmed by SNP-A. (c) CN-neutral LOH as determined using the SNP array. In the upper histogram red dots represent the log 2 intensity ratio for each SNP locus and the blue line below it shows the averaged log 2 values. The corresponding chromosome idiogram and location of heterozygous SNP calls (small green vertical bars) are shown. Pink and blue bars below the idiogram indicate areas of LOH. MS genotyping and CN confirmation using real-time PCR is shown. Black bars demonstrate the DNA CN in both bone marrow and normal T (CD3 þ ) cells obtained from the same patient. MS ID marker is displayed above the bars. Y axis (RQ) presents the relative quantification scale where 1 equals diploid DNA CN. Normal karyogram of chromosome 6 is also included. CN, copy number; LOH, loss of heterozygosity; MC, metaphase cytogenetics; MDS, myelodysplastic syndromes ; MS, microsatellite; SNP-A, single nucleotide polymorphism DNA arrays.
affected by deletions as demonstrated by traditional cytogenetics, including 7q and 11q. Particularly informative were newly detected lesions that occurred in an overlapping fashion in multiple patients. As expected, certain chromosomes were more often affected, including loss of chromosomal material within chromosomes 1, 5, 7 and 11, or gain of chromosome 8. Leukemia
Other interesting defects not previously described in the context of MDS included changes on chromosome 1p31.1 (N ¼ 6), add(2)(q37) (N ¼ 6) and del(14)(q11) (N ¼ 5). The results presented in this letter show that SNP-A analysis in MDS will allow for detection of known and previously cryptic lesions. The resulting increased detection rate is due to the
Letters to the Editor
2061 improved resolution of the genomic scan, while its sensitivity remains comparable to that of MC. As a result, the proportion of patients with normal karyotype decreased compared to routine MC, and more patients showed multiple lesions. It is likely that the presence of additional changes as detected by SNP-A analysis is responsible for the variability of the clinical phenotype associated with a known karyotype. Of significant interest is that regions of UPD detected by SNP-A were also located in portions of chromosomes frequently affected by genomic losses. We suggest that SNP-A constitutes a major advance in comparison to MS-based identification of LOH. Theoretically, the resolution of SNP-A is limited only by the number of SNP probes used, while its sensitivity depends on the contribution of the dysplastic clone to the overall cellular content of the sample. One major advantage of SNP-A is the ability to perform analyses on interphase cells without the need for cell division. This advantage is reflected by the low proportion of non-informative results. Unlike traditional MC, SNP-A do not allow for the distinction of multiple clones and minor clones may remain undetected due the ‘dilution’ effect. In general, SNP-A showed very good correlation with MC. However, in a proportion of patients lesions detected by MC were not found by SNP-A. This discrepancy is due to various reasons, including the fact that loss of Y is not tested on the array and balanced translocations and inversions cannot be detected by this technique. Finally, the remaining discrepancy is due to lower sensitivity of SNP-A for smaller clones. Our analysis was performed using total bone marrow. Sorted myeloid cells were not available but it is likely that lymphocyte depletion would result in a higher detection rate. Despite this shortcoming, clonal lesions were detected and the fact that the defects were seen despite the contamination suggests that the clones detected were of significant size. While this clearly decreases the sensitivity, it may be an advantage as only large likely clinically significant clones were studied. In sum, we have written this letter as we believe that SNP-Abased karyotyping is a technology that should be further explored as it may complement metaphase karyotyping in improved prediction of clinical phenotype and prognosis in the diagnosis of MDS and allow for better mapping of the breakpoints. To our knowledge, our letter describes the first systematic application of this technology in MDS. We hope that
we can inspire further investigations and discussion on innovative ways to improve cytogenetic diagnosis of MDS.
LP Gondek1, R Tiu1, AS Haddad1, CL O’Keefe1, MA Sekeres2, KS Theil3 and JP Maciejewski1,2 1 Experimental Hematology and Hematopoiesis Section, Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA; 2 Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, USA and 3 Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH, USA E-mail:
[email protected]
References 1 Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89: 2079–2088. 2 Sole F, Luno E, Sanzo C, Espinet B, Sanz GF, Cervera J et al. Identification of novel cytogenetic markers with prognostic significance in a series of 968 patients with primary myelodysplastic syndromes. Haematologica 2005; 90: 1168–1178. 3 List AF, Vardiman J, Issa JP, DeWitte TM. Myelodysplastic Syndromes. Hematology 2004; 2004: 297–317. 4 Mori N, Morosetti R, Mizoguchi H, Koeffler HP. Progression of myelodysplastic syndrome: allelic loss on chromosomal arm 1p. Br J Haematol 2003; 122: 226–230. 5 Maeck L, Haase D, Schoch C, Hiddemann W, Alves F. Genetic instability in myelodysplastic syndrome: detection of microsatellite instability and loss of heterozygosity in bone marrow samples with karyotype alterations. Br J Haematol 2000; 109: 842–846. 6 Cervantes RB, Stringer JR, Shao C, Tischfield JA, Stambrook PJ. Embryonic stem cells and somatic cells differ in mutation frequency and type. Proc Natl Acad Sci USA 2002; 99: 3586–3590. 7 Raghavan M, Lillington DM, Skoulakis S, Debernardi S, Chaplin T, Foot NJ et al. Genome-wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic recombination in acute myeloid leukemias. Cancer Res 2005; 65: 375–378. 8 Slater HR, Bailey DK, Ren H, Cao M, Bell K, Nasioulas S et al. High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs. Am J Hum Genet 2005; 77: 709–726.
Elevated mRNA transcripts of non-homologous end-joining genes in pediatric acute lymphoblastic leukemia Leukemia (2007) 21, 2061–2064; doi:10.1038/sj.leu.2404742; published online 10 May 2007
Human cells are frequently exposed to DNA double-strand breaks (DSB) that are generated either by endogenous (for example, V(D)J recombination and reactive oxygen species) or by exogenous (for example, ionizing radiation and chemotherapy regimens) agents. To repair DSB and maintain genome integrity, human cells have evolved two major repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR uses homologous DNA strand as a template to repair DSB and is an error-free process.1 NHEJ repairs DSB by
directly re-ligating DNA ends, which may be error-prone and may produce mis-ligation between the two chromosome ends.2–4 In this regard, it has been shown that NHEJ repair is involved in the generation of certain chromosomal translocations in acute myeloid and lymphoid leukemia.2,3,5 These chromosome translocations are thought to be correlated with highly activated NHEJ activity.2,3 However, the expressions of NHEJ genes in these tumors are poorly understood so far. In this study, we report a survey of NHEJ mRNA expressions in pediatric acute lymphoblastic leukemia (ALL). To investigate a panel of NHEJ gene expressions in pediatric ALL, we examined mRNA transcripts of all NHEJ family members, including Ku70, Ku80, DNA–PK, Artemis, XRCC4, Leukemia
