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Originally published as JCO Early Release 10.1200/JCO.2007.11.2649 on October 29 2007 © 2007 American Society of Clinical Oncology. Mutation Status of the Residual ATM Allele Is an Important Determinant of the Cellular Response to Chemotherapy and Survival in Patients With Chronic Lymphocytic Leukemia Containing an 11q Deletion
From the Cancer Research UK Institute for Cancer Studies, University of Birmingham; Department of Epidemiology and Public Health, University of Birmingham, Birmingham; Haematology Department, Royal Bournemouth Hospital, Bournemouth; Medical Research Council Toxicology Unit, Leicester University, Leicester, United Kingdom; and Institute for Human Genetics, University Hospital Schleswig-Holstein, Kiel, Germany Address reprint requests to Tatjana Stankovic, CRUK Institute for Cancer Studies, Vincent Dr, University of Birmingham, Birmingham B15 2TT, United Kingdom; e-mail: t.stankovic{at}bham.ac.uk
Purpose The ataxia telangiectasia mutated (ATM) gene is located on chromosome 11q and loss of this region is common in B-cell chronic lymphocytic leukemia (CLL). Our aim was to determine if CLL tumors with a chromosome 11q deletion might be divided into two subgroups based on the status of the remaining ATM allele. Methods The sequence of the residual ATM allele was determined in 72 CLLs with an 11q deletion. This was related to the cellular response to irradiation or cytotoxic drug exposure in vitro and clinical outcome. Results We show that the residual ATM allele is mutated in 36% of CLLs with an 11q deletion and that these leukemias demonstrate an impaired cellular response to irradiation or cytotoxic drug exposure in vitro. Inactivation of the second ATM allele was associated with a reduction in patient survival beyond that already dictated by the presence of an 11q deletion (P = .0283). Furthermore, we demonstrate that ATM mutations may arise during the evolution of an 11q deleted subclone and are associated with its expansion. Conclusion CLL with 11q deletion can be divided into two subgroups based on the integrity of the residual ATM allele. Patients with complete loss of ATM function, due to biallelic ATM defects, have defective responses to cytotoxic chemotherapeutics in vitro and a poorer clinical outcome. ATM mutant subclones can develop during an individual's disease course and give rise to additional expansion of the 11q deleted subclone.
B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by remarkable clinical heterogeneity.1 A deletion of the long arm of chromosome 11 is found in 10% to 20% of CLL patients and identifies a group with a poor outcome.1-5 The deletion includes the ataxia telangiectasia mutated (ATM) gene located at 11q22.3 to q23.1.6 ATM is mutated in a substantial minority of CLL patients, and the presence of an ATM mutation has itself been shown to confer impaired survival.7-11 The ATM protein is a central component of the signal transduction pathway that is activated in response to DNA double-strand breaks (DSBs) and synchronizes DNA repair with the induction of p53-dependent apoptosis.12 Individuals who inherit two germline ATM mutations develop the disorder ataxia telangiectasia, characterized by progressive neurodegeneration and predisposition for the development of lymphoid malignancies.13 The cellular consequences of ATM dysfunction include chromosomal radiosensitivity, radioresistant DNA synthesis, loss of cell cycle check points, and p53 dysfunction.12 p53 activity is pivotal for induction of apoptosis in response to agents that cause DNA damage in CLL cells.14-16 Thus, impairment in the activation of p53 is associated with a poor prognosis in CLL patients and may occur either directly through TP53 gene mutation or indirectly through ATM inactivation.17,18 In contrast to the distinct cellular phenotype of biallelic ATM inactivation, the significance of loss of a single ATM allele is less clear. Acquired heterozygous mutations of ATM have been reported in CLL and found to be associated with impaired responses to DNA damage. However, this observation could be explained by a dominant negative effect of some mutant ATM proteins or by haploinsufficiency.9,19 To date, the relationship among the presence of chromosome 11q deletions, ATM mutations, and the DNA damage response pathway has not been addressed comprehensively in CLL. The aim of this study was to determine whether there is a differential impact of mono- or biallelic ATM defects on activation of the ATM pathway in response to chemotherapy, and whether inactivation of the remaining ATM allele in CLL patients with an 11q deletion has an impact on tumor progression and survival beyond that influenced by the deletion itself.
Patient Samples We investigated a cohort of 72 CLL patients in whom 11q deletion was identified by fluorescent in situ hybridization (FISH; LSI ATM probe, Vysis; Abbott Diagnostics, Abbott Park, IL). Ethical approval was obtained from South Birmingham Ethics Committee. The patients had been diagnosed with CLL during 28 years and had been managed in Birmingham, Bournemouth, or Leicester hospitals according to United Kingdom national guidelines.20 We included all of the known patients who had been found to have a chromosome 11q deletion affecting more than 5% of the tumor clone and had available stored tumor material. The samples had been collected either at the patients’ diagnosis or at variable times during follow-up. In 19 patients, samples were available from two time points during the natural history of the individual's disease; in the 53 patients, samples were available from a single time point. Outcome measures were analyzed on the basis of disease status on September 31, 2006. The ATM mutation status was not available to treating physicians at the time of management decisions.
Mutation Analysis
Treatment With Ionizing Irradiation and Chemotherapeutics
Western Blotting
Statistics
ATM Mutations Develop in More Than One Third of CLL Tumors With a Chromosome 11q Deletion We identified the presence of an ATM mutation in a leukemia sample from 26 (36%) of the 72 patients with an 11q deletion (Table 1; Appendix Table A1, online only24-30). Among the 19 patients with consecutive chronological samples, six had an ATM mutation that was present in both early and later samples, and nine had a remaining wild-type ATM allele in both samples. There were no CLL tumors that exhibited loss of an ATM mutation over time. However, in four of 19 patients an ATM mutation was absent from the first sample but present in the later follow-up sample, indicating that it had developed during an individual's disease progression (Fig 1). Thus, we distinguished two genetic subgroups of CLL patients with a deletion of chromosome 11q: those who acquired a mutation in the residual ATM allele during disease ontology and those in whom the residual ATM allele remained wild type.
ATM Mutations Affect DNA Damage Responses to Irradiation in CLLs With Chromosome 11q Deletions ATM is a protein kinase that responds to DNA DSBs.12 We investigated the impact of an additional ATM mutation in CLLs with an 11q deletion on in vitro ATM-dependent signaling induced by IR. Through expansion of our previous analyses, we confirmed differential ATM kinase activity according to the status of the residual ATM allele.9 We demonstrated preservation of IR-induced ATM autophosphorylation and SMC1 trans-phosphorylation by ATM in 11 of 14 and 11 of 11 leukemias, respectively, with a retained wild-type ATM allele. By comparison, there was a highly significant impairment of phosphorylation of both respective proteins in 13 of 14 and 12 of 13 leukemias with an ATM mutation (Figs 2 and 3; Table 2; Appendix Table A2, online only).
The downstream consequences of ATM-dependent phosphorylation of p53 include p53 upregulation and transcriptional activation of p21.17 We showed that eight of eight leukemias with one retained ATM allele exhibited IR-induced p53 phosphorylation and p21 induction. By comparison, seven of eight and seven of nine leukemias with biallelic ATM defects exhibited reduced p53 phosphorylation and impaired p21 responses, respectively. Differences in the levels of IR-induced phosphorylation of ATM targets and p21 expression were significant (Figs 3 and 4; Table 2; Appendix Table A2).
The size of the 11q-deleted subclone varied between patients. Among leukemias with biallelic ATM defects, there was no correlation between the size of 11q-deleted subclone and cellular responses. However, in the majority of these leukemias studied, a high percentage (> 60%) of the CLL clone carried the 11q deletion. Interestingly, a similar lack of correlation was observed in the ATM wild-type tumors despite considerable variability in 11q-deleted subclone size (Appendix Tables A2 and A3, online only). Together, our results suggest that, within the sensitivity levels of our experiments, the presence of one wild-type ATM allele is sufficient for normal ATM activity. Finally, we tested whether the presence of an additional ATM mutation in 11q-deleted leukemias influences IR-induced cellular killing. We demonstrated significantly reduced killing of CLL cells among 10 leukemias with an ATM mutation compared with 10 leukemias with wild-type ATM by measuring the percentage of viable cells (mean 36% v 77%; P = .003) and PARP1 cleavage at 24 hours after IR (Appendix Fig A1, online only).9 Our data confirm differential activation of ATM-dependent signaling and cellular killing in response to IR between ATM mutant and wild-type CLLs with 11q deletions. Phosphorylation of ATM and SMC1 provided the best stratification between the two genetic subsets.
ATM Mutations Determine In Vitro Cellular Responses to Chemotherapeutic Drugs in 11q-Deleted CLL Tumors
ATM Mutations Affect the Clinical Outcome of CLL Patients With a Chromosome 11q Deletion We next investigated whether the differential in vitro responses translated into differences in clinical outcome between the two genetic subgroups. First, we compared survival from diagnosis in patients with a second mutant or wild-type ATM allele. Patients with an ATM mutant had a significantly shorter survival time compared with those with no mutation (median, 83 v 134 months; P = .0283; Fig 7). By comparison, the presence of an ATM mutation was not significantly associated with a shorter treatment-free survival (P = .421; Table 2).
Because of the retrospective nature of sample collection, we considered whether there could be an influence of differential sampling between these two 11q-deleted subgroups. However, the median time from diagnosis to the time of ATM analysis was similar between ATM mutant and wild-type patients (P = .228), suggesting that selective bias imposed by sampling was unlikely to account for differences in survival between the two subgroups (Appendix Table A4, online only). There was also a trend for a shorter survival of patients with an additional ATM mutation as measured from the date of the ATM mutation analysis (median, 21.1 v 49.2 months; P = .0715; Appendix Fig A4). The absence of a significant difference here could relate to the shorter follow-up time (median, 17 months). We addressed whether the impact of ATM status on survival from diagnosis was independent of other prognostic features using Cox regression and comparing unadjusted and adjusted hazard ratios (HRs; relative risk of death in patients with ATM-mutated compared with wild-type ATM). Notably, patients with an ATM mutation in addition to an 11q deletion were found to be more than twice as likely to die compared with those patients with 11q-deleted CLLs with a residual wild-type ATM allele (unadjusted HR 2.55; 95% CI, 1.12 to 5.81), and this HR was not significantly altered after adjustment for IGVH status (adjusted HR, 2.54; 95% CI, 1.11 to 5.80). Similarly, there were no significant alterations on the HR of ATM status after adjustment for age (HRunadj = 2.34; HRadj = 2.19), Zap70 (HRunadj = 2.87; HRadj = 2.19), and 17p deletion status (HRunadj = 2.20; HRadj = 2.18).
In a previous report the prognostic impact of 11q deletions was only observed in younger CLL patients.3 In contrast, we found that the effect of ATM mutation on survival was similar in the age groups Finally, we observed no associations between the presence of an ATM mutation and other prognostic indicators, including clinical stage (P = .788), IgVH status (P = .536), CD38 expression (P = .182), Zap70 expression (P = .270), or the presence of a chromosome 17p deletion (P = .251; Table 2). In summary, ATM mutation appears to be an independent predictor of survival in this cohort of patients with 11q-deleted CLL.
ATM Mutations Are Associated With Expansion of the 11q-Deleted Subclone Within the CLL Population
We performed serial FISH on four CLLs with late development of an ATM mutation. Notably, in 3/4 leukemias the expansion of the 11q deleted subclone was coincident with the occurrence of a mutation in the residual ATM allele (Fig 8B). These results suggest that the development of an ATM mutation favors an expansion of the subclone carrying the 11q deletion. Lack of systematic cytogenetic analysis prevented us from assessing the impact of ATM mutation on wider genetic instability. However, FISH analysis for detection of chromosome 6q, 13q, and 17p deletions, trisomy of chromosome 12, and rearrangements of immune system genes in 18 wild-type and 10 ATM mutant 11q-deleted CLLs revealed no differences in the frequency of these abnormalities between the two subgroups (P = .68).
We have investigated the prevalence and consequences of an additional ATM mutation in CLLs with chromosome 11q deletion, a defect that itself leads to loss of one ATM allele.6 We have shown that 11q-deleted CLLs can be divided into two genetic subgroups depending on the status of the remaining ATM allele. These subgroups exhibit differential cellular responses to irradiation and chemotherapy-induced DNA damage, which translate into an impact on disease progression and patient survival. Several studies have confirmed that a chromosome 11q deletion is associated with impaired clinical outcome in CLL patients.2-5 In our cohort of 72 patients, all with an 11q deletion, we found that the second ATM allele was mutated in 36% of patients. Furthermore, those patients who had developed an ATM mutation had a poorer survival from diagnosis than those in whom the residual ATM allele was wild type. These data suggest that loss of one or two functional ATM alleles might have an additive effect that directly influences patient survival. Interestingly, the prevalence of ATM mutations in this chromosome 11q–deleted CLL cohort is three-fold higher than the 12% frequency that we observed in unselected CLL tumors.9 Thus, the heterozygous ATM status seems to be a risk factor for the formation and maintenance of ATM mutant subclones. We found direct evidence for the development of an ATM mutation on an 11q-deleted background during disease progression in four tumors. The late acquisition of chromosome 11q deletions has also been described.33 Therefore, our findings suggest that the loss of function of each ATM allele could occur in a stepwise manner. As such, leukemias with biallelic ATM abnormalities might represent a more advanced disease stage than those with a single allelic deletion or mutation. The ATM protein plays a central role in coordination of a rapid cellular response to DNA DSBs.9,12 One consequence of impaired ATM activity is dysfunctional p53 activation after IR.17 We found that only 11q-deleted tumors with a second mutant ATM allele exhibited this dysfunctional response. Interestingly, differential DNA damage–induced p53 responses have also been reported previously among 11q-deleted CLL tumors using a fluorescent activated cell–based assay.34,35 We suggest that these patterns are also likely to relate to the mutation status of the remaining ATM allele. Given that activation of p53 after DNA damage is critical for the induction of apoptosis, it is significant that we were able to demonstrate relative resistance to IR-induced cellular killing among the 11q-deleted leukemias with ATM mutations.16,17 We showed that the chemotherapeutics used as first-line therapy in CLL (fludarabine, cyclophosphamide, and chlorambucil) induce DNA DSBs in CLLs in an analogous manner to IR and can activate ATM-dependent signaling leading to differential responses among the two subgroups of 11q-deleted tumors.12,20,31 A degree of redundancy is known to exist between ATM and related proteins such as ATR in the activation of the DNA damage response.36 However, our results indicate that these alternative kinases do not compensate for the loss of ATM signaling in response to either IR or drugs in CLL.36 This is consistent with the finding that ATR activity is downregulated in noncycling CLL cells.37 Our observations suggest that acquisition of a second allelic ATM defect promotes disease progression through the functional loss of the ATM-dependent DNA-damage response and preferential cellular survival. We provided additional support for this concept by showing a significant association of an ATM mutation with an increased size of 11q-deleted subclone and the coincidental expansion of the 11q subclone with the development of an ATM mutation. It is conceivable that DNA damaging agents in CLL with a monoallelic ATM loss could potentially provide a selective pressure for the emergence and expansion of subclones with biallelic ATM defect. Indeed, two of the patients with late acquisition of an ATM mutation received chemotherapy before the development of the mutation. Loss of ATM function is unlikely to be the only abnormality determining poor outcome of patients with a chromosome 11q deletion.38,39 Notably, more than 60% of leukemias in our cohort did not have an ATM mutation. Therefore, expansion of these subclones is likely to rely on alternative survival mechanisms. The minimal region of 11q deletion includes a number of genes, and although no mutations have been identified in alternative candidates, epigenetic gene silencing and haplo-insufficiency at certain loci might contribute to the aggressive phenotype of 11q-deleted CLL with wild-type ATM.6,38,39 We believe that this study significantly extends understanding of the biology of CLL with an 11q deletion. We have shown that CLL patients with this chromosome defect are at risk of developing a mutation in the remaining ATM allele, and this in turn leads to more rapid clonal expansion and reduced survival. We suggest that the use of agents that bypass the ATM/p53 apoptotic pathway might be beneficial for these patients.40-44
The author(s) indicated no potential conflicts of interest.
Conception and design: Belinda Austen, David Oscier, A. Malcolm Taylor, Paul A. Moss, Tatjana Stankovic Provision of study materials or patients: Belinda Austen, David Oscier, Martin Dyer, Paul A. Moss Collection and assembly of data: Belinda Austen, Anna Skowronska, Claire Baker, Judith E. Powell, Anne Gardiner, David Oscier, Aneela Majid, Martin Dyer, Tatjana Stankovic Data analysis and interpretation: Belinda Austen, Anna Skowronska, Claire Baker, Judith E. Powell, Reiner Siebert, Tatjana Stankovic Manuscript writing: Belinda Austen, A. Malcolm Taylor, Paul A. Moss, Tatjana Stankovic Final approval of manuscript: Belinda Austen, Anna Skowronska, Claire Baker, Judith E. Powell, Anne Gardiner, David Oscier, Martin Dyer, A. Malcolm Taylor, Paul A. Moss, Tatjana Stankovic
We thank Graham Fews, Claire Almond, and Paul Biggs for their technical assistance, Victoria Weston, PhD, for useful comments, and the Leukaemia Research Fund UK for its continuous support.
published online ahead of print at www.jco.org on October 29, 2007. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Byrd JC, Gribben JG, Peterson BL, et al: Select high-risk genetic features predict earlier progression following chemoimmunotherapy with fludarabine and rituximab in chronic lymphocytic leukemia: Justification for risk-adapted therapy. J Clin Oncol 24:437-443, 2006 2. Döhner H, Stilgenbauer S, Benner A, et al: Genomic aberrations and survival in chronic lymphocytic leukaemia. N Engl J Med 343:1910-1916, 2000 3. Döhner H, Stilgenbauer S, James MR, et al: 11q deletions identify a new subset of B-cell chronic lymphocytic leukaemia characterised by extensive nodal involvement and inferior prognosis. Blood 89:2516-2522, 1997 4. Fegan C, Robinson H, Thompson P, et al: Karyotypic evolution in CLL: Identification of a new sub-group of patients with deletions of 11q and advanced or progressive disease. Leukaemia 9:2003-2008, 1995[Medline] 5. 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Stankovic T, Stewart G, Fegan C, et al: Ataxia telangiectasia mutated-deficient B-cell chronic lymphocytic leukaemia occurs in pregerminal center cells and results in defective damage response and unrepaired chromosome damage. Blood 99:300-309, 2002 11. Schaffner C, Stilgenbauer S, Rappold G, et al: Somatic ATM mutations indicate a pathogenic role of ATM in B-cell chronic lymphocytic leukaemia. Blood 94:748-753, 1999 12. Lobrich M, Jeggo P: Harmonising the response to DSBs: A new string in the ATM bow. DNA Repair 4:749-759, 2005[Medline] 13. Taylor AM, Byrd PJ: Molecular pathology of ataxia telangiectasia. J Clin Pathol 58:1009-1015, 2005 14. Rao VA, Plunkett W: Activation of a p53-mediated apoptotic pathway in quiescent lymphocytes after the inhibition of DNA repair by fludarabine. Clin Cancer Res 9:3204-3212, 2003 15. Rosenwald A, Chuang EY, Davis RE, et al: Fludarabine treatment of patients with chronic lymphocytic leukaemia induces a p53-dependent gene expression response. Blood 104:1428-1434, 2004 16. Stankovic T, Hubank M, Cronin D, et al: Microarray analysis reveals that TP53- and ATM-mutant B-CLLs share a defect in activating proapoptotic responses after DNA damage but are distinguished by major differences in activating prosurvival responses. Blood 103:291-300, 2004 17. Pettitt AR, Sherrington PD, Stewart G, et al: p53 dysfunction in B-cell chronic lymphocytic leukaemia: Inactivation of ATM as an alternative to TP53 mutation. Blood 98:814-822, 2001 18. Lin K, Sherrington PD, Dennis M, et al: Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia. Blood 100:1404-1409, 2002 19. Scott SP, Bendix R, Chen P, et al: Missense mutations but not allelic variants alter the function of ATM by dominant interference in patients with breast cancer. Proc Natl Acad Sci USA 99:925-930, 2002 20. 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Alvi AJ, Austen B, Weston VJ, et al: A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down regulation of genes involved in transcription regulation and survival. Blood 105:4484-4491, 2005 Submitted February 13, 2007; accepted September 6, 2007.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
H2AX) were detected using a standard immunofluorescence technique
2 test); IGVH, Zap70, CD38, and 17p status; cellular killing; cytogenetic abnormalities (Fisher's exact test); size of 11q-deleted subclone; time to analysis (Mann-Whitney U test); and cellular responses to IR and drugs (Mann-Whitney U test, t test); and also between 11q-deleted clone size and cellular responses (Spearman's rank correlation). 
65 and younger than 65 years (HR65+ = 2.15; 95% CI, 0.84 to 5.5; HR< 65 = 1.95; 95% CI, 0.48 to 7.8). 
