Dec 23, 2013 - Chromosome 11 architecture, 11q23 minimally deleted region and BIRC3 deletion ..... mutation and much sup
ARTICLES
Chronic Lymphocytic Leukemia
ATM mutation rather than BIRC3 deletion and/or mutation predicts reduced survival in 11q-deleted chronic lymphocytic leukemia: data from the UK LRF CLL4 trial Matthew J.J. Rose-Zerilli,1 Jade Forster,1 Helen Parker,1 Anton Parker,2 Ana E. Rodríguez,3 Tracy Chaplin,4 Anne Gardiner,2 Andrew J. Steele,1 Andrew Collins,5 Bryan D. Young,4 Anna Skowronska,6 Daniel Catovsky,7 Tatjana Stankovic,6 David G. Oscier,1,2 and Jonathan C. Strefford1
Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; 2Department of Haematology, Royal Bournemouth Hospital, Bournemouth, UK; 3Cancer Research Center (IBMCC-CSIC), University of Salamanca, Spain; 4Centre for Medical Oncology, Barts and The London School of Medicine and Dentistry, London, UK; 5Human Development and Health, Faculty of Medicine, University of Southampton, UK; 6School of Cancer Sciences, University of Birmingham, UK; and 7Haemato-oncology Research Unit, Division of Molecular Pathology, The Institute for Cancer Research, Sutton, UK 1
ABSTRACT
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ATM mutation and BIRC3 deletion and/or mutation have independently been shown to have prognostic significance in chronic lymphocytic leukemia. However, the relative clinical importance of these abnormalities in patients with a deletion of 11q encompassing the ATM gene has not been established. We screened a cohort of 166 patients enriched for 11q-deletions for ATM mutations and BIRC3 deletion and mutation and determined the overall and progression-free survival among the 133 of these cases treated within the UK LRF CLL4 trial. SNP6.0 profiling demonstrated that BIRC3 deletion occurred in 83% of 11q-deleted cases and always co-existed with ATM deletion. For the first time we have demonstrated that 40% of BIRC3-deleted cases have concomitant deletion and mutation of ATM. While BIRC3 mutations were rare, they exclusively occurred with BIRC3 deletion and a wildtype residual ATM allele. In 11q-deleted cases, we confirmed that ATM mutation was associated with a reduced overall and progression-free survival comparable to that seen with TP53 abnormalities, whereas BIRC3 deletion and/or mutation had no impact on overall and progression-free survival. In conclusion, in 11q-deleted patients treated with first-line chemotherapy, ATM mutation rather than BIRC3 deletion and/or mutation identifies a subgroup with a poorer outcome.
Introduction
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Deletion of chromosome 11q (termed del11q) was first recognized as a recurrent karyotypic abnormality acquired during the course of the disease in patients with progressive chronic lymphocytic leukemia (CLL).1 Subsequent interphase fluorescence in situ hybridization (FISH) analysis identified 11q deletion in approximately 20% of patients with CLL, and associations with bulky lymphadenopathy and a poorer outcome for patients under the age of 55 years were noted.2,3 Subsequent studies have documented associations with unmutated IGHV genes, del13q, genomic complexity, short telomeres, progressive disease and a poor outcome in response to alkylating agent or purine analog treatment, which was improved by their use in combination and ameliorated by the further addition of rituximab.4–10 Genomic profiling studies have refined previous karyotypic and FISH studies showing that 11q deletions are mono-allelic, frequently large and include a minimally deleted region (MDR), which encompasses the ATM gene.11–14 Evidence that ATM is a key target of 11q deletions is derived from findings that: 1) mutation of the ATM gene is found in 30-40% of patients with an 11q deletion;15,16 2) the presence of an ATM mutation results in impaired DNA damage responses;15,17–19
and 3) patients in the UK LRF CLL4 trial with biallelic ATM abnormalities (deletion and mutation) have a poorer outcome following the initial therapy with alkylating agent and/or purine analog therapy compared to those with mono-allelic ATM deletion or mutation.20 However, uncertainty remains as to whether the poorer outcome of patients with 11q deletion in the absence of an ATM mutation is simply a consequence of ATM haploinsufficiency. Alternative possibilities that have been considered include deletion, mutation or epigenetic silencing of other genes either within or outside the MDR or the associated genomic complexity.8,21–23 Neither candidate gene sequencing nor whole exome sequencing studies have identified mutations within other genes located in the MDR.24,25 However, recent data have revealed a high incidence of deletion or, more rarely, mutation of BIRC3, a negative regulator of noncanonical NFκB signaling located at 11q22. It has been reported that BIRC3 deletion and/or mutation (previously termed “BIRC3 disruption”) occurs in a mutually exclusive manner with TP53 abnormalities, is associated with fludarabine-resistance,26 and when detected at diagnosis predicts for poor overall survival independent of 11q deletion.27 The above data strongly suggest that there are subsets of del11q patients that exhibit differing responses to standard
©2014 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2013.098574 The online version of this article has a Supplementary Appendix. Manuscript received on September 27, 2013. Manuscript accepted on December 23, 2013. Correspondence:
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haematologica | 2014; 99(4)
11q23 deletions, ATM and BIRC3 mutations in CLL
treatment. However, the relative frequency and clinical significance of ATM and BIRC3 abnormalities is unclear. This study addresses this issue in a large cohort of 11qdeleted patients detected by SNP6 profiling and screened for ATM and BIRC3 mutations. As a consequence, in the context of a phase III clinical trial of chemotherapy, we show that ATM mutational status remains the most clinically informative genomic lesion in 11q-deleted CLL, identifying cases with outcome comparable to TP53 deletion and/or mutation patients, and that the presence of BIRC3 deletion and/or mutation is associated with outcome comparable to other 11q-deleted CLL cases.
made. An additional 33 11q-deleted patients were also included to allow more significant associations between the 11q deletion and other genomic variables to be made, such as with mutational status of ATM and BIRC3. The additional del11q patients were sampled subsequent to the development of progressive disease, with a median time from diagnosis of 3.2±5.2 years (±1 standard deviation (SD); range 1 month-27 years). For the entire cohort of 166 patients, mutational data were available for TP53 (n=125), SF3B1 (n=140) and NOTCH1 (n=146).28,29 Details on the molecular diagnostic assays30,31 are available in the Online Supplementary Methods. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki and our local ethics committee gave their approval for the study.
Methods DNA extraction, SNP6 array hybridization, data extraction and analysis
A total of 166 untreated CLL patients diagnosed according to standard morphological and immunophenotypic criteria were included in this study (Table 1 and Online Supplementary Table S1). This cohort principally included patients from the UK LRF CLL4 trial9 (n=133) which allowed accurate clinical correlations to be
Genomic DNA was extracted from CLL B cells (n=166) and buccal swabs (n=32), prior to being purified, amplified, labeled and hybridized to the Affymetrix SNP6.0 platform (Affymetrix, Santa Clara, CA, USA) as previously described32 (Online Supplementary Methods).
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Patients and molecular diagnostic assays
Characteristics
Sub-groups
All Cases (%)
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Table 1. Cohort characteristics.
CLL4 Cases n (%)2 del(11q)
97 (73) 71 (73) 26 (27) 71 56-86 23 (24) 47 (49) 27 (28) 9 (9) 88 (91) 51 (53) 46 (47) 9 (9) 88 (91) 56 (64) 32 (36) 32 (42) 44 (58) 42 (51) 40 (49) 5 (10) 8 (17) 35 (73) 7 (8) 76 (92) 0 (0) 97 (100) 0 (0) 95 (100) 10 (12) 75 (88) 19 (24) 61 (76)
36 (27) 27 (75) 9 (25) 62 44-80 7 (19) 18 (50) 11 (31) 3 (8) 33 (92) 22 (61) 14 (39) 1 (3) 35 (97) 30 (83)1 6 (17) 18 (55) 15 (45) 21 (64) 12 (36) 10 (32)3 4 (13)4 17 (55) 2 (6) 30 (94) 32 (89) 4 (11) 2 (6) 34 (94) 2 (6) 32 (94) 3 (9) 29 (91)
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Trisomy 12 by SNP6
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13q by SNP6
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17p by SNP6
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IGHV mutation statusa CD38 positivityb
166 (100) 124 (75) 42 (25) 63 39-89 48 (31) 69 (44) 40 (25) 13 (8) 153 (92) 94 (66) 72 (44) 12 (7) 154 (93) 98 (68) 47 (32) 68 (50) 69 (50) 74 (54) 63 (46) 17 (16) 19 (18) 69 (66) 9 (7) 116 (93) 57 (34) 109 (66) 3 (2) 159 (98) 14 (10) 132 (90) 28 (20) 112 (80)
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Age at diagnosis
Male Female Mean Range A B C Trisomy Normal Deleted Normal Deleted Normal Unmutated Mutated + + T-Mutated NT-Mutated Unmutated Mutated Unmutated Deleted Normal Mutated Unmutated Mutated Unmutated Mutated Unmutated
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Number of cases Gender
ZAP70 positivityc ATM mutation statusd
TP53 mutation statuse BIRC3 deletion status BIRC3 mutation status NOTCH1 mutation statusf SF3B1 mutation statusg
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non-del(11q)
A proportion of cases were not screened for our panel of molecular and cytogenetic biomarkers; not screened= 21 a, 29 b, 29 c, 61 d, 41 e, 20 f, 26 g. 1A significant positive association was identified between the presence of del11q and an unmutated IGHV sequences in the CLL4 cases (P=0.03, 2x2 χ2 test). 2The columns for del11q and non-del11q are based on the SNP6.0 profiling data. 3A significant positive association was identified between the frequency of truncating ATM mutations (T-Mutated) and presence of del11q. (P=0.02, 2x2 χ2 test). 4The frequency of non-truncating ATM mutations (NT-Mutated) was similar in non-del11q and del11q cases. (P=0.76, 2x2 χ2 test).
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M. J.J. Rose-Zerilli et al.
The presence or absence of somatically-acquired single nucleotide variants (SNVs or mutations) in ATM and BIRC3 were successfully ascertained in 105 (CLL4 cases: n=79 of 133) and 162 (CLL4 cases: n=131 of 133) patients, respectively. Denaturing high-performance liquid chromatography (DHPLC), high-resolution melt (HRM) polymerase chain reaction (PCR) analysis and Sanger sequencing were utilized.20,26,27,29 More details of methods and the strategy adopted for assigning the somatic nature of each SNV are provided in the Online Supplementary Methods and Online Supplementary Table S2.
Statistical analysis Statistical analysis was performed using SPSS (v.20). Associations with treatment response and clinical outcome were only performed on the LRF UK CLL4 cases due to the homogeneous and well-annotated nature of this cohort (Online Supplementary Methods). The LRF UK CLL4 cohort included in
this current study did not differ significantly from the entire cohort for an extensive panel of variables, with the enrichment exception of del11q (P=0.002) and del13q (P
