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Mar 1, 2011 - Texas MD Anderson Cancer Center, Houston, TX, USA. E-mail: ... NRASmut in a large cohort of children with AML who were treated on two ...

Letters to the Editor 4

The Molecular Diagnostics Laboratory, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. E-mail: [email protected] 5 These authors contributed equally to this work.

References 1 Vajdic CM, van Leeuwen MT. What types of cancers are associated with immune suppression in HIV? Lessons from solid organ transplant recipients. Curr Opin HIV AIDS 2009; 4: 35–41. 2 Law GR. Invited commentary: do clusters of leukemia and lymphoma provide evidence for an infectious cause? Am J Epidemiol 2005; 162: 823–824. 3 Ben-Bassat I, Raanani P, Gale RP. Graft-versus-leukemia in chronic lymphocytic leukemia. Bone Marrow Transplant 2007; 39: 441–446. 4 Ritgen M, Bottcher S, Stilgenbauer S, Bunjes D, Schubert J, Cohen S et al. Quantitative MRD monitoring identifies distinct GVL response patterns after allogeneic stem cell transplantation for chronic lymphocytic leukemia: results from the GCLLSG CLL3X trial. Leukemia 2008; 22: 1377–1386. 5 Krackhardt AM, Witzens M, Harig S, Hodi FS, Zauls AJ, Chessia M et al. Identification of tumor-associated antigens

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in chronic lymphocytic leukemia by SEREX. Blood 2002; 100: 2123–2131. Khouri IF, Saliba RM, Admirand J, O’Brien S, Lee MS, Korbling M et al. Graft-versus-leukaemia effect after non-myeloablative haematopoietic transplantation can overcome the unfavourable expression of ZAP-70 in refractory chronic lymphocytic leukaemia. Br J Haematol 2007; 137: 355–363. Penn DJ, Damjanovich K, Potts WK. MHC heterozygosity confers a selective advantage against multiple-strain infections. Proc Natl Acad Sci USA 2002; 99: 11260–11264. Thursz MR, Thomas HC, Greenwood BM, Hill AV. Heterozygote advantage for HLA class-II type in hepatitis B virus infection. Nat Genet 1997; 17: 11–12. Doherty PC, Zinkernagel RM. Enhanced immunological surveillance in mice heterozygous at the H-2 gene complex. Nature 1975; 256: 50–52. Therodorou I, Abel L, Mauro F, Duprey B, Magnac C, PayelleBrogard B et al. High occurrence of DRB1 11 in chronic lymphocytic leukaemia families. Br J Haematol 2002; 119: 713–715. Hojjat-Farsangi M, Jeddi-Tehrani M, Amirzargar A, Razavi SM, Sharifian RA, Rabbani H et al. Human leukocyte antigen class II allele association to disease progression in Iranian patients with chronic lymphocytic leukemia. Hum Immunol 2008; 69: 666–674.

Prevalence and clinical implications of NRAS mutations in childhood AML: a report from the Children’s Oncology Group Leukemia (2011) 25, 1039–1042; doi:10.1038/leu.2011.31; published online 1 March 2011

Over the last several years, molecular analyses in acute myeloid leukemia (AML) have established clinical–pathological and prognostic correlations that have better defined subgroups in this heterogeneous disease and influenced risk-adapted therapeutic approaches on recent clinical trials. Genetic markers such as FLT3 internal tandem duplications (FLT3-ITD), were first identified in adults1 and subsequently in children,2 as conferring a poor prognosis. By contrast, mutations in nucleophosmin (NPM1)3,4 and more recently, CCAAT/enhancer-binding protein alpha (CEBPA)5 have been found to be associated with improved outcomes in both adult and pediatric studies. However, despite being initially described almost 20 years ago, the prognostic implications of mutations involving the RAS proto-oncogenes, remain controversial.6 The RAS gene family is comprised of three homologues, HRAS (11p15.5), KRAS (12p12.1) and NRAS (1p13.2), all of which function as membrane-associated guanosine nucleotide phosphate-binding proteins.7 Mutations in RAS genes are frequent in AML and serve as prototypic Class I lesions, initiating key downstream hyperproliferative signal transduction pathways.8 NRAS mutations (NRASmut) are the most common, occurring in 10–20% of AML patients. RAS mutations have been linked to leukemic progression in myelodysplastic syndrome and an inferior outcome in AML by some researchers;9–13 whereas other studies have reported no survival difference in AML patients harboring RAS mutations compared with those without RAS mutations.14,15 Still other reports have suggested the presence of a RAS mutation may actually improve survival and/or response to therapy.15 Here, we report on the incidence and prognostic significance of NRASmut in a large cohort of children with AML who were treated on two recent cooperative group studies.

Of the 1241 eligible patients with de novo AML enrolled on CCG-2961 and COG-AAML03P1, diagnostic DNA was available for 825 children. Demographics, laboratory and clinical characteristics, and outcome for patients with and without available specimens were comparable to the study population at large, as reviewed in previous studies.7 KRAS mutations were examined for 183 patients treated on CCG-2961, and only two mutations were identified, so no further analysis was conducted. HRAS mutations are rare in AML,15 and thus not examined in this study (data not shown). Exons 1 and 2 of the NRAS gene were amplified, sequenced and evaluated for genomic alterations. Of the 825 specimens tested, 86 (10%) had a missense mutation in the amplified coding region. Mutations involved either codon 12 (n ¼ 52, 60%) or 13 (n ¼ 33, 38%), as have been previously described, with one unique mutation in codon 29. No mutations were identified in codon 61, a third codon previously reported to be involved in adult AML (Supplementary Table 1).16 In order to establish the somatic, disease-associated nature of these mutations, DNA extracted from remission specimens available from 12 patients with NRASmuts was evaluated for the presence of NRASmuts. We failed to demonstrate the presence of NRASmuts in the remission specimens, demonstrating that the NRASmuts were diseaseassociated mutations and not germline polymorphisms. Sex, age, and race were comparable between NRASmut positive and wild-type patients. Similarly, there was no difference in frequency of organomegaly or extramedullary disease (central nervous system involvement or chloroma). Median diagnostic WBC (28.9  103/ml vs 21.6  103/ml, P ¼ 0.19) and platelet counts (45  103/ml vs 48  103/ml, P ¼ 0.97) at diagnosis were also similar between the two cohorts. Diagnostic bone marrow blast counts were lower in patients with NRASmut than in those without the mutation (64% vs 71%, P ¼ 0.05) (Supplementary Table 1). NRAS mutational status was correlated with the French–American–British Classification of AML subtypes17 for Leukemia

Letters to the Editor

1040 historic comparisons. AML French–American–British Classification subgroups were similarly distributed between patients with and without NRASmut, with 30% having M2 or M4 morphology (Figure 1a). The presence of NRASmut was correlated with specific cytogenetic and molecular alterations in our study population. NRASmut were similarly distributed among the major cytogenetic groups (Figure 1b). Cytogenetic information was available on 563/901 (62.5%) of the de novo patients treated on CCG2961. On COG-AAML03P1, 316/340 (93%) of patients had available cytogenetic data of which 185/316 (59%) were centrally reviewed. Of the 86 NRASmut patients, 68 had cytogenetic information available for further comparisons, of whom 17 (25%) had a normal karyotype and another 17 (25%) had abnormalities of chromosome 11q23. These frequencies were comparable to those found with wild-type NRAS. Deletions or monosomies of chromosome 7 [-7/del(7q)] were rare in patients with NRASmut (3%) and no patient with NRASmut had monosomy 5 or del (5q) (Figure 1b). NRASmut has been previously found to correlate with abnormalities of chromosomes 3 [inv(3)/t(3;3), t(3;5)]15,16 and 16 [inv(16)/ t(16;16)], 16 however, this was not found in our study. Seven percent of NRASmut were associated with t(6;9)(p23;q24) compared with only 2% of wild-type NRAS (P ¼ 0.014). However, the presence of NRASmut had no impact on the poor prognosis of patients with t(6;9)(p23;q24), who demonstrated 3-year overall survival (OS) of 0% vs 38±34% (P ¼ 0.696) and event-free survival (EFS) of 0% vs 16±32% (P ¼ 0.986) for NRASmut and wild-type patients, respectively. t(6;9)(p23;q24) translocations were frequently associated with the poor prognostic marker FLT3-ITD, even in NRASmut patients (2/5 patients with t(6;9) and NRASmut; 7/9 with t(6;9) and wild-type NRAS), in which FLT3-ITD was uncommon (see below). Correlation of NRASmut with common mutations in AML was also determined. FLT3-ITD appeared to be less common in those with NRASmut (7 vs 13%, P ¼ 0.115); whereas NPM1 mutations were significantly more common in those with NRASmut (16 vs 6%, P ¼ 0.003) compared with wild type. There was no association observed between NRASmut and CEBPA or WT1 mutations (Figure 1c). Five patients with NRASmut and WT1 mutations had a third abnormality (t(8;21), 2 patients; inv(16), 2 patients and t(9;11), 1 patient). Overall, 77 of 86 patients with NRASmut (90%) had at least one additional mutation or cytogenetic abnormality (Supplementary Figure 1). NRASmut status was correlated with specific risk group. NRASmut was more prevalent in those with low-risk AML, (P ¼ 0.028) as defined by the presence of the core-binding factor (CBF) group (t(8;21)(q22;q22) or inv(16)t(16;16)), CEBPA or NPM1 mutations (NPMc þ ). However, this association appeared to be in large part because of the significant association

of NRASmuts with NPMc þ . There was no significant correlation with individual CBF abnormalities, with comparable numbers of patients demonstrating t(8;21)(q22;q22) with and without NRASmut (16 vs 15%, P ¼ 0.80), and similarly for abnormalities of chromosome 16 (13 vs 11%, P ¼ 0.63). In contrast, NRASmut was inversely related with high-risk disease; only 4% of those with NRASmut had monosomy 7, monosomy 5/del(5q) or FLT3-ITD, with a high allelic ratio compared with 15% of patients without the mutation (P ¼ 0.007, Supplementary Table 1). Response to induction chemotherapy and clinical outcome was assessed for patients with and without NRASmut. Patients with NRASmut had similar complete remission (CR) rates after one course of induction chemotherapy as patients without the mutation (85 vs 80%, P ¼ 0.268). The actuarial OS at 5 years from study entry was 47±13% for the NRASmut patients compared with that of 55±4%, for those without the mutation (P ¼ 0.364, Supplementary Figure 2a), with a corresponding EFS of 38±11% and 44±4% (P ¼ 0.55, Supplementary Figure 2b) for those with and without NRASmut, respectively. Those in CR had a similar relapse risk, regardless of the presence of NRASmut (39±12 vs 36±4%, P ¼ 0.973, Supplementary Figure 2c) with an actuarial disease-free survival rate of 44±13% vs 52±4%, P ¼ 0.257, respectively (Supplementary Figure 2d). As NRASmut were more prevalent in patients with low-risk disease, we considered whether NRASmut might have a clinical impact in specific risk groups in AML. The presence of NRASmut did not affect clinical outcome in patients with low-risk disease; the OS rate from study entry for those with NRASmut was 59±20 vs 73±6% for those without NRASmut, P ¼ 0.114. For those who achieved an initial CR, the relapse risk was 25±16 vs 24±7% for those with and without NRASmut, P ¼ 0.956, with a corresponding disease-free survival rate of 52±19 vs 61±8%, P ¼ 0.190. Next, we examined whether NRASmut could delineate prognosis in two heterogenous subgroups, specifically, patients with standard-risk AML and those with normal karyotypes. Within the standard-risk group, NRASmut had no effect on the OS rate (42±20% vs 51±6%, P ¼ 0.669), EFS rate (31±17 vs 40±6%, P ¼ 0.692), relapse risk (44±21 vs 42±7%, P ¼ 0.712) or disease-free survival rate (41±20 vs 51±7%, P ¼ 0.616). Similarly, the OS rate (24±35 vs 55±9%, P ¼ 0.356), EFS rate (24±25 vs 45±10%, P ¼ 0.230), relapse risk (47±27 vs 39±10%, P ¼ 0.707) and disease-free survival rate (28±24% vs 51±11%, P ¼ 0.106) were comparable between patients with normal karyotypes with or without the mutation (Supplementary Table 2). As there were only 3 patients with high-risk disease who carried an NRASmut, meaningful clinical assessment of NRASmut could not be performed for this cohort, but all 3 patients with NRASmut and other high-risk features either failed to achieve remission (N ¼ 1) or

Figure 1 Morphologic and genetic correlations of NRASmuts in childhood AML. (a) Distribution of French–American–British AML subtype of pediatric patients who underwent NRAS mutational analysis. (b) Distribution of cytogenetic abnormalities of patients with and without NRASmut. (c) Distribution of mutations in FLT3-ITD, NPM, CEBPA and WT1 genes in patients with and without NRASmut. nDenotes statistically significant difference. Leukemia

Letters to the Editor

1041 had a relapse after achieving an initial CR (N ¼ 2), with no survivors. This current retrospective study represents an evaluation of a large pediatric cohort specifically focused on the incidence and impact of NRASmut on outcome. We identified NRASmuts in 10% of children with AML treated on two consecutive cooperative clinical trials. NRASmut were found predominantly at codon 12 in keeping with other reports15,16 and also at codon 13, although none were present at codon 61, a third commonly mutated location. Bacher et al.16 identified codon 61 mutations specifically in adult AML samples with inv(16)/t(16;16) or inv(3)/ t(3;3) and found these cytogenetic abnormalities were overrepresented in the NRASmut positive population. A high frequency of NRASmut in AML samples harboring inv(16)/ t(16;16) has also been observed in other adult studies.15,18 We did not find a higher frequency of abnormalities of chromosome 16 between patients with NRASmut and those with wild-type NRAS (P ¼ 0.63), which is consistent with our previous data2 and likely accounts for the absence of codon 61 abnormalities. By extension, the higher frequency of low-risk AML that we observed in NRASmut patients cannot be attributed to a higher frequency in CBF AML as seen in adults, but rather appears to be a result of an association with NPM1 mutations. NRASmut is a proto-typical Class I ‘proliferation/survival lesion’, which has been proposed to require collaboration with Class II ‘differentiation/self-renewal’ lesions to induce leukemogenesis.7,8,19 CBF abnormalities represent Class II lesions. CEBPA mutations, are also good-risk Class II lesions that are frequently found in normal karyotype AML.3 However, we observed no relationship between CEBPA mutations and NRASmut, which is consistent with adult data.19 We did, however, detect a significantly higher frequency of NRASmut in patients with NPM1 mutations (P ¼ 0.0003). NPM1 mutations represent another good-risk Class II lesion. This correlation has not been previously described in adults,19 however, it appeared responsible for the association with low-risk AML that we observed. The presence of more than one Class I lesion is uncommon in AML,19 thus, it is in keeping with previous studies15 that NRASmut and the high-risk FLT3-ITD were rarely found together in the same patient. Other high-risk features such as monosomy 5 or 7 or del (5q) were similarly uncommon in NRASmut patients. Taken together, these data suggest that this dual class paradigm of leukemogenesis remains valid in NRAS-mediated AML, although collaboration with distinct Class II abnormalities may distinguish adult and pediatric disease. NRASmut did not affect the outcome in pediatric AML, as we found similar rates of CR and relapse, as well as 5-year EFS and OS rates in patients with the mutated and wild-type NRAS. These findings were consistent even when only patients with standard-risk or normal karyotype disease were considered. Although NRASmut may not provide prognostic information in AML, the role of this mutation in activation of the RAS/MEK/ERK signal transduction pathway is well documented20 and provides a key regulatory step in the transformation of myeloid progenitors and the evolution of AML. Murine studies have demonstrated that interrupting the RAS signal transduction pathway may induce cellular apoptosis in AML cells harboring the NRASmut, and thus, interruption of the signaling pathway may have clinical utility.21 Risk-adapted therapy on the basis of prognostic implications of recurrent molecular lesions represents the most significant advance in both adult and pediatric AML management in recent years. Pediatric AML remains a unique subset of myeloid diseases that are distinct from their adult counterparts and

worthy of independent evaluation. Our study has demonstrated similarities with that of Bowen et al.15 regarding the lack of association of NRASmut with FLT3-ITD, but also highlighted differences with a novel association with NPM1 mutations, t(6;9) translocations and absence of association with CBF leukemias. Given lack of clinical significance for NRASmuts, implementation of NRASmuts for risk-based therapy allocation in pediatric AML cannot be justified at this time. However, as this mutation remains an attractive target for directed therapy, NRASmut profiling should be performed and documented as part of correlative research protocols.

Conflict of interest Dr Craig Hurwitz is employed by Reatta Pharmaceuticals and Dr Janet Franklin is employed by Amgen Pharmaceuticals. The other authors declare no conflict of interest.

Acknowledgements AAML03P1 and CCG 2961 are funded by grants U10 CA98543 and U10 CA98413. Myeloid Leukemia Reference Laboratory is supported by U10 CA114766.

JN Berman1,2, RB Gerbing2, TA Alonzo2,3, PA Ho4,5, K Miller4, C Hurwitz6, NA Heerema7, B Hirsch8, SC Raimondi9, B Lange10, JL Franklin11, A Gamis12 and S Meshinchi2,4,5 1 Department of Pediatrics, IWK Health Centre and Dalhousie University, Halifax, Nova Scotia, Canada; 2 Children’s Oncology Group, Arcadia, CA, USA; 3 Department of Biostatistics, University of Southern California, Los Angeles, CA, USA; 4 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 5 Department of Pediatrics, University of Washington, Seattle, WA, USA. 6 Reata Pharmaceuticals, Dallas, TX, USA; 7 Department of Pathology, The Ohio State University, Columbus, OH, USA; 8 Department of Laboratory Medicine and Pathology, University of Minnesota Cancer Center, Minneapolis, MN, USA; 9 Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA; 10 Department of Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 11 Amgen Pharmaceuticals, Thousand Oaks, CA, USA and 12 Division of Hematology/Oncology/Bone Marrow Transplantation, Children0 s Mercy Hospital & Clinics, Kansas City, MO, USA E-mail: [email protected]

References 1 Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002; 99: 4326–4335. 2 Zwaan CM, Meshinchi S, Radich JP, Veerman AJ, Huismans DR, Munske L et al. FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance. Blood 2003; 102: 2387–2394. Leukemia

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1042 3 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. 4 Cazzaniga G, Dell’Oro MG, Mecucci C, Giarin E, Masetti R, Rossi V et al. Nucleophosmin mutations in childhood acute myelogenous leukemia with normal karyotype. Blood 2005; 106: 1419–1422. 5 Ho PA, Alonzo TA, Gerbing RB, Pollard J, Stirewalt DL, Hurwitz C et al. Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children’s Oncology Group. Blood 2009; 113: 6558–6566. 6 Janssen JW, Steenvoorden AC, Lyons J, Anger B, Bohlke JU, Bos JL et al. RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes. Proc Natl Acad Sci USA 1987; 84: 9228–9232. 7 Frohling S, Scholl C, Gilliland DG, Levine RL. Genetics of myeloid malignancies: pathogenetic and clinical implications. J Clin Oncol 2005; 23: 6285–6295. 8 Gilliland DG, Tallman MS. Focus on acute leukemias. Cancer Cell 2002; 1: 417–420. 9 Hirai H, Kobayashi Y, Mano H, Hagiwara K, Maru Y, Omine M et al. A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome. Nature 1987; 327: 430–432. 10 Hirai H, Okada M, Mizoguchi H, Mano H, Kobayashi Y, Nishida J et al. Relationship between an activated N-ras oncogene and chromosomal abnormality during leukemic progression from myelodysplastic syndrome. Blood 1988; 71: 256–258. 11 Lubbert M, Mirro Jr J, Kitchingman G, McCormick F, Mertelsmann R, Herrmann F et al. Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia. Oncogene 1992; 7: 263–268. 12 Misawa S, Horiike S, Kaneko H, Sasai Y, Ueda Y, Nakao M et al. Significance of chromosomal alterations and mutations of the N-RAS and TP53 genes in relation to leukemogenesis of acute myeloid leukemia. Leuk Res 1998; 22: 631–637.

13 Paquette RL, Landaw EM, Pierre RV, Kahan J, Lubbert M, Lazcano O et al. N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome. Blood 1993; 82: 590–599. 14 Nakagawa T, Saitoh S, Imoto S, Itoh M, Tsutsumi M, Hikiji K et al. Multiple point mutation of N-ras and K-ras oncogenes in myelodysplastic syndrome and acute myelogenous leukemia. Oncology 1992; 49: 114–122. 15 Bowen DT, Frew ME, Hills R, Gale RE, Wheatley K, Groves MJ et al. RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years. Blood 2005; 106: 2113–2119. 16 Bacher U, Haferlach T, Schoch C, Kern W, Schnittger S. Implications of NRAS mutations in AML: a study of 2502 patients. Blood 2006; 107: 3847–3853. 17 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR et al. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 1985; 103: 620–625. 18 Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, Auvrignon A et al. Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia 2006; 20: 965–970. 19 Schlenk RF, Dohner K, Krauter J, Frohling S, Corbacioglu A, Bullinger L et al. Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia. N Engl J Med 2008; 358: 1909–1918. 20 Zuber J, Tchernitsa OI, Hinzmann B, Schmitz AC, Grips M, Hellriegel M et al. A genome-wide survey of RAS transformation targets. Nat Genet 2000; 24: 144–152. 21 Kim WI, Matise I, Diers MD, Largaespada DA. RAS oncogene suppression induces apoptosis followed by more differentiated and less myelosuppressive disease upon relapse of acute myeloid leukemia. Blood 2009; 113: 1086–1096.

Supplementary Information accompanies the paper on the Leukemia website (

Microarray detection of multiple recurring submicroscopic chromosomal aberrations in pediatric T-cell acute lymphoblastic leukemia Leukemia (2011) 25, 1042–1046; doi:10.1038/leu.2011.33; published online 8 March 2011

Cytogenetic studies have an important role in the diagnosis and prognosis of hematological malignancies, but the conventional karyotype analysis in acute lymphoblastic leukemia (ALL) is often hampered by suboptimal chromosome morphology, low lymphoblast mitotic activity, and inability to detect subtle (o5–10 Mb) abnormalities. With the combination of karyotype analysis and fluorescence in situ hybridization (FISH), cytogenetic studies of chromosome aberrations have become a major and reliable marker for risk assignment and risk-directed treatment of pediatric B-cell ALL.1 In contrast to B-cell ALL, T-cell ALL (T-ALL) is associated with few unique cytogenetic features on which to base risk-group stratification. Recently, a few genome-wide microarray studies have revealed submicroscopic chromosome aberrations in T-ALL.2–4 To identify submicroscopic chromosome aberrations in pediatric patients with a well-defined diagnosis of T-ALL and to determine the feasibility of incorporating a newly developed 133K-targeted oligonucleotide-based microarray platform into routine cytogenetic diagnosis, we used this comprehensive system to analyze DNA from cryopreserved diagnostic leukemia samples and compared microarray findings to their reported bone marrow karyotypes. Multiple submicroscopic abnormalities in each of Leukemia

these samples have been revealed with many recurrent abnormalities. This study included 22 leukemia bone marrow samples from the Children’s Oncology Group (COG) Cell Bank. These specimens were collected, with informed consent for future research use and institutional review board approval, from children with T-ALL who were treated on COG ALL clinical trials. Mononuclear cells were purified by Ficoll-Hypaque centrifugation prior to cryopreservation. All specimens consisted of 490% lymphoblasts. Samples were selected based on the presence of an identified deletion or rearrangement involving chromosome 6q (originally designed to validate the microarray analysis). The T-ALL diagnosis and the diagnostic karyotypes received COG central review. Oligonucleotide-based microarray analysis was performed using a custom-designed 133K-feature whole-genome microarray (Signature OncoChip, manufactured by RocheNimbleGen, Madison, WI, USA). The custom-designed array targeted 41800 genes and genomic intervals associated with cancer at a probe spacing ranging from B1 probe per 0.2–7.0 kb. In addition, the genomic backbone of the array had an average spacing of B1 probe per 35 kb. Array comparative genomic hybridization analysis was performed as described previously.5 Karyotypes of the 22 patient samples included the following: deletion in 6q (n ¼ 11), deletion in 6q with additional abnormalities (n ¼ 7), and apparently normal (n ¼ 4) (Table 1).

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