ALBERT

Description: Analytical Chemistry DOI: 10.1021/ac3025416

Description: Failure of initial remission-induction therapy and disease recurrence remains a major problem in the management of acute leukemia, however the nature of the biologic factors promoting relapse are incompletely understood. To identify genomic abnormalities associated with relapse, we performed high-resolution, genome-wide analysis of DNA copy number abnormalities and loss-of heterozygosity using Affymetrix single nucleotide polymorphism (SNP) microarrays in 33 cases of relapsed acute leukemia. Sixteen ALL (2 ETV6-RUNX1, 2 MLL rearranged, 6 pseudodiploid or cytogenetically normal B-progenitor ALL, and 6 T-lineage) and 17 AML (two RUNX1-RUNX1T1 [AML1-ETO], two MLL-rearranged, one M7, and 12 with normal karyotype or miscellaneous cytogenetic abnormalities) were studied. Samples with less than 80% blasts were flow sorted to at least 90% purity prior to DNA extraction. Diagnostic samples were available for all cases, and germline samples for 22. DNA copy number and LOH analysis was performed using Affymetrix 250k Nsp and Sty arrays was performed for all samples. Data were analysed using a karyotype-guided normalization algorithm, dChipSNP, and circular binary segmentation. In a detailed comparative analysis of paired diagnostic and relapse samples, changes in DNA copy number abnormalities were identified in the relapse sample in 14 of 16 (87.5%) ALL cases. A striking finding was loss of copy number lesions present at diagnosis in 8/16 ALL relapse samples, and the acquisition of new copy number lesions in 4 of these 8 samples. In each case, the pattern of deletions at antigen receptor loci was comparable between relapse and diagnosis, suggesting the emergence of a related leukemic clone, rather than the development of a distinct second leukemia. An additional 8 ALL relapse samples retained the copy number lesions present at diagnosis, but 6 of these acquired additional copy number abnormalities at relapse, a finding further suggestive of significant clonal evolution. Of the newly acquired copy number abnormalities in the relapse samples, deletions (62.5% of cases) were more common than gains (12.5%). In constrast to ALL a more restricted range of copy number abnormalities was seen in AML, with new abnormalities at relapse seen in 5/17 (29.4%) cases, and deletions (29.4%) outnumbering gains (17.6%). The loss of lesions present at diagnosis was only observed in two AML relapse samples. Examining the entire cohort, the CDKN2A/B locus was most commonly involved (N=5), gains of 1q were noted in two cases, otherwise all observed copy number changes were noted in single cases only, and included focal deletions of ERG and RUNX1. Copy neutral loss of heterozygosity was uncommon, with the exception of three AML cases with UPD of the entire chromosome 13. These observations indicate that relapse is frequently the result of the emergence of a leukemic clone that shows significant genetic differences from the diagnostic clone. Whether these represent rare clones present at the time of diagnosis or are the emergence of new clones as the result of ongoing genomic abnormalities can now be determined using genomic probes specific for the newly acquired deletions.

Description: Expression of BCR-ABL1 is the hallmark of chronic myelogenous leukemia (CML) and a subset of de novo acute lymphoblastic leukemia (ALL), but the factors determining disease lineage, and progression of CML to myeloid or lymphoid blast crisis, are incompletely understood. We recently reported deletion of IKZF1 (encoding the lymphoid transcription factor Ikaros) in 85% of de novo pediatric and adult BCR-ABL1 ALL, and in lymphoid blast crisis in a small cohort of CML cases (Nature2008;453:110), suggesting that IKZF1 deletion is important in the pathogenesis of BCR-ABL1 lymphoid leukemia. To identify genetic determinants of disease stage and blast crisis lineage in CML, we have now performed high-resolution, genome wide analysis of DNA copy number abnormalities (CNA) and loss-of heterozygosity (LOH) and candidate gene resequencing in a cohort of 90 CML patients that included 64 samples obtained at chronic phase (CP), 15 samples at accelerated phase (AP), 9 lymphoid blast crisis (LBC) and 22 myeloid blast crisis (MBC) samples. Importantly, 25 patients had sequential samples (CP and/or AP, as well as blast crisis samples) enabling analysis of lesions acquired at progression to blast crisis. All blast crisis samples were flow sorted to at least 90% purity prior to DNA extraction. Germline samples for 28 cases obtained at remission or by flow sorting of blast crisis samples were also examined. Affymetrix SNP 6.0 arrays, interrogating over 1.87 million genomic loci, were used for 85 samples, and 500K arrays for the remainder. Identification of tumor-specific (somatic) copy number analysis was performed by directly comparing CML samples to matched germline samples were available, or by filtering results against databases of inherited copy number variants for samples lacking germline material. Genomic resequencing of IKZF1, PAX5 and TP53 was performed for all AP, LBC and MBC samples. There were few CNAs in CP-CML (mean 0.27 deletions and 0.07 gains per case), with no recurring lesions identified apart from deletions or gains at the chromosomal breakpoints of BCR and ABL1 (3 cases each). Notably, the size of these translocation associated deletions was highly variable, ranging from 6kb (one ABL1 deletion) and 15 kb (one BCR deletion) to deletions extending to the telomeres of chromosomes 9 and 22. No significant increase in lesion frequency was identified in AP cases (0.14 deletions and 0.9 gains per case), however the number and cumulative extent of genomic aberrations was significantly higher in both lymphoid and myeloid blast crisis samples. LBC cases had a mean of 8.1 deletions/case (P

Description: To identify cooperating lesions in core-binding factor acute myeloid leukemia, we performed single-nucleotide polymorphism-array analysis on 300 diagnostic and 41 relapse adult and pediatric leukemia samples. We identified a mean of 1.28 copy number alterations per case at diagnosis in both patient populations. Recurrent minimally deleted regions (MDRs) were identified at 7q36.1 (7.7%), 9q21.32 (5%), 11p13 (2.3%), and 17q11.2 (2%). Approximately one-half of the 7q deletions were detectable only by single-nucleotide polymorphism-array analysis because of their limited size. Sequence analysis of MLL3, contained within the 7q36.1 MDR, in 46 diagnostic samples revealed one truncating mutation in a leukemia lacking a 7q deletion. Recurrent focal gains were identified at 8q24.21 (4.7%) and 11q25 (1.7%), both containing a single noncoding RNA. Recurrent regions of copy-neutral loss-of-heterozygosity were identified at 1p (1%), 4q (0.7%), and 19p (0.7%), with known mutated cancer genes present in the minimally altered region of 1p (NRAS) and 4q (TET2). Analysis of relapse samples identified recurrent MDRs at 3q13.31 (12.2%), 5q (4.9%), and 17p (4.9%), with the 3q13.31 region containing only LSAMP, a putative tumor suppressor. Determining the role of these lesions in leukemogenesis and drug resistance should provide important insights into core-binding factor acute myeloid leukemia.

Description: In genome-wide analyses of DNA copy number abnormalities (CNAs) and loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) microarrays, we previously reported a high frequency of genetic alterations of regulators of B lymphoid development and cell cycle in B-progenitor acute lymphoblastic leukemia (B-ALL), and a near-obligate deletion of the transcription factor Ikaros (IKZF1) in BCR-ABL1 ALL (Nature2007;446:758 and Nature2008;453:110). To assess the prognostic significance of genetic alterations and their effects on the leukemic cell transcriptome, we have now extended these studies to a cohort of 221 NCI high risk pediatric ALL cases treated on the Children’s Oncology Group (COG) P9906 trial. The majority of these cases (N=170) lacked known cytogenetic abnormalities, and none were BCR-ABL1 positive. CNA and LOH data were generated using Affymetrix 500K SNP arrays, and gene expression data was available for 207 cases. Genomic resequencing was performed for PAX5 and IKZF1. These analyses revealed mutations in genes regulating B lymphoid development in 57.5% of cases, including PAX5 CNAs (31.7%), PAX5 sequence mutations (8.1%), IKZF1 deletions (24.9%) and IKZF1 sequence mutations (2.6%). In addition, recurring CNAs were detected in a number of other genes known to play roles in transformation and drug response including CDKN2A/B (45.7%), RB1 (11.3%), BTG1 (10.4%), IL3RA (6.8%), KRAS (6.3%), NRAS (2%), NR3C1 (2%), NR3C2 (3%), and ERG (6.3%). To examine associations between genetic alterations and outcome in a genome-wide fashion, we used a semi-supervised principal components approach and identified a predictor of outcome driven by deletion/mutation of IKZF1, EBF, and BTLA (hazard ratio 3.74, P=8.1×10−5). IKZF1 lesions were most strongly associated with poor outcome (cumulative incidence of relapse 68.8% in IKZF1-mutated ALL vs. 29.1% for IKZF1 wild type cases, P=0.002). This copy number predictor also predicted outcome in an independent cohort of 258 B-progenitor ALL cases treated at St Jude Children’s Research Hospital (HR 3.76, P=0.00023). Importantly, the St Jude cohort had a much higher proportion of cases with conventional recurring cytogenetic abnormalities including hyperdiploidy and translocations than the COG P9906 cohort (60.4% versus 23%, respectively), including BCR-ABL1 (N=21; 8.1%). Notably, the association of IKZF1 mutation and poor outcome was also observed in the cases lacking BCR-ABL1. The presence of an IKZF1 mutation was also associated with a high-level of minimal residual disease following induction therapy in both cohorts. We next defined a gene expression profile of high risk (IKZF1-mutated) ALL in each cohort. Using gene set enrichment analysis (GSEA), we found the high risk gene expression signature to be positively enriched for hematopoietic stem cell genes and genes associated with minimal residual disease, and negatively enriched for genes regulating cell cycle progression and genes upregulated during B cell maturation. Moreover, GSEA also demonstrated significant similarity of the expression signatures of high risk COG and St Jude ALL, a striking finding given the marked differences in sample composition of the two groups. As IKZF1 mutation is a near obligate lesion in BCR-ABL1 lymphoid leukemia (Nature2008;453:110) and in this study is a strong independent predictor of poor outcome in BCR-ABL1 negative ALL, we next examined similarity of the gene expression signatures of high risk (IKZF1-mutated) BCR-ABL1 negative ALL and BCR-ABL1 positive ALL. Using GSEA and direct comparison of differentially expressed genes, there was highly significant similarity between the two signatures. These findings indicate that IKZF1 mutation is a key determinant of the gene expression signature of both BCR-ABL1 positive and negative ALL, and is thus likely to be central to the pathogenesis and poor outcome of both leukemia subtypes. Thus, IKZF1 deletion is a new prognostic factor in ALL, and is associated with a gene expression profile indicative of impaired maturation of the leukemic blasts, an observation compatible with the known role of IKZF1 in regulating early lymphoid development. These findings have important implications for risk stratification of ALL.

Description: Abstract 50 Genomic aberrations are important prognostic factors in chronic lymphocytic leukemia (CLL) [Döhner et al., 2000]. However, known genomic aberrations fail to fully explain the biologic and clinical heterogeneity of the disease. We sought to precisely map copy number alterations (CNA) and copy number neutral losses of heterozygocity (LOH) to better characterize known recurrent aberrations and to identify new genetic lesions. We used Affymetrix 6.0 single nucleotide polymorphism (SNP) array analyses on CD19 sorted CLL cells. Data were analyzed using dChipSNP, a modified array normalization algorithm guided by cytogenetic abnormalities and a circular binary segmentation. We studied samples from 346 patients enrolled on the CLL8 trial of the German CLL Study Group. Data of 145 samples were analyzed against intraindividual reference DNA (paired), data of 201 samples against a pool of reference DNA (unpaired). FISH data were available for all samples, the distribution of genomic aberrations was as follows: del(13q14) in 59.8%, del(11q23) in 26.3%, trisomy 12 in 11.6%, and del(17p13) in 8.4%. IGHV was mutated in 32.9%, unmutated in 63.3%, and unknown in 3.8%. In total, 261 tumor-specific CNA could be discovered among the 145 paired samples. Genomic aberrations were found in 85.5% of these cases. The average number of aberrations per case was 1.8; according to the hierarchical model of genomic aberrations, it was 3.5 in del(17p), 2.4 in del(11q23), 1.7 in del(13q14) single, and 0.5 in normal karyotype CLL. The minimally deleted region (MDR) on 13q14 was 277.25 kb in size and contained mir15a and mir16, DLEU1 and DLEU2; RFP2 was not part of the MDR. Deletions on 13q were highly heterogeneous in size, ranging from 294 kb to 68 Mb. On 11q23 the MDR only contained ATM, the smallest lesion of 78.5 kb being intragenic; in two of theses cases, the deletion size was too small to be detected by FISH analysis. TP53 was affected in all del(17p13) cases except two; one tumor-specific deletion of 635.7 kb was detected in cytoband 17p13.2 harboring 30 genes and a second deletion of 780 kb in 17p13.3 containing – among 15 other genes – MNT, a tumor suppressor acting as an antagonist of MYC. A partial trisomy on chromosome 12 was not detected. Of the 261 CNA, 95 were located in genomic regions that are not evaluated by our routine FISH probe panel; 17 regions were affected recurrently: del(1p35.3) [2/145], del(1q23.3) [2/145], del(1q42.12) [2/145], +2p [5/145], del(3p21.31) [2/145], del(6p25.3) [3/145], +(6p25.3) [2/145], del(6q) [11/145], del(7q23.1) [2/145], +(8q24.21) [3/145], del(9q13-q21.13) [2/145], del(10q24) [2/145], del(14q24.3) [2/145], del(14q12.3) [2/145], del(15q15.1) [2/145], +18 [3/145] and +19 [7/145]. The frequency of these CNA was subsequently evaluated within the cohort of 201 (unpaired) samples. Five of 17 regions were affected in more than 2% in the whole cohort: +2p, del(6q), +8q24.21, del(15q15.1), and +19. Gain of 2p was found in 6.9% of cases, the minimally amplified region was 1.9 Mb in size and contained e.g. BCL11A and REL. Del(6q) was detected in 6,4%, the deletions were heterogeneous, an MDR could not be identified. 16 cases had 8q24.21 gains, the minimally amplified region was delineated by three intragenic gains in MYC. 14 cases had loss in 15q15.1 focussing on MGA, a potential suppressor of transcriptional activation by MYC. 8 cases had total or partial gains of chromosome 19, among those two overlapping partial gains with a minimally amplified region of 2.17 Mb in 19p13.2. Tumor-specific LOH were identified in 6.0% (9/145) located on 13q in three cases and in one case each on 17p, 12q, 11p, 1p, 3 and 22q. The LOH on chromosomes one and three overlapped with recurrent losses in 1p35.3 and 3p21.31. Essential members of the ATR-pathway were located in these regions: ATRIP and RPA2. However, mutational analyses of the two candidate genes in 48 cases revealed no mutations. SNP array analysis is a reliable tool to identify and further characterize genomic aberrations in CLL. MDR on 13q14 was delineated to a 277.25 kb segment affecting mir15a, mir16, DLEU1 and DLEU2 but not RFP2; the MDR on 11q23 to a segment only containing ATM. Cases with del(11q23) and del(17p) showed a higher genomic complexity than those with normal karyotype or del(13q14) as single abnormality. Relatively few novel genetic lesions were identified. Although occurring at low frequency, they may lead to the discovery of new genes involved in CLL pathogenesis. Disclosures: Stilgenbauer: Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

Description: Abstract 849 Core-binding-factor (CBF) acute myeloid leukemia (AML) defined by the presence of t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is associated with favorable outcome. However, about 30–40% of patients are not cured by current treatment approaches. Secondary genetic changes are believed to cause clinical heterogeneity. To identify new secondary genetic lesions, we performed high-resolution, genome-wide analysis of copy number aberrations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 300 adult and pediatric CBF AMLs; t(8;21), n=157 (adult, n=114; pediatric, n=43); and inv(16), n=143 (adult, n=104; pediatric, n=39). Germline control DNA from remission bone marrow or peripheral blood was available for paired analysis in 175 patients. In addition, for 42 patients matched relapse samples were analyzed. Data were processed using reference alignment, dChipSNP and circular binary segmentation. Paired analysis revealed a median of 1.28 somatic CNAs per case [t(8;21): 1.14, range: 0–5; inv(16): 1.45, range: 0–9], with deletions more common than gains [t(8;21): 0.94 losses/case vs. 0.2 gains/case; inv(16): 0.95 vs. 0.5]. Recurrent deletions were detected at chromosomal bands 7q36.1 (n=23), 9q21.13 (n=15), 11p13 (n=7), 17q11.2 (n=6), 10q24.32 (n=2), and at the chromosomal breakpoints of t(8;21) and inv(16) on 8q21.3 (n=9), 21q22 (n=16), 16p13.11 (n=28), and 16q22.11 (n=21). Deletions at 7q, 11p and 17q were validated using FISH analysis. Minimally deleted regions (MDR) less than 1.5 Mb were identified at 7q36.1 (647 Kb, 4 genes), 9q21.13 (1125 Kb, 9 genes), 11p13 (130 Kb, 1 gene), and 17q11.2 (902 Kb, 11 genes), with each region containing a putative tumor suppressor (e.g., MLL3 on 7q, FRMD3 on 9q, WT1 on 11p, and NF1 on 17q). Sequence analysis of MLL3 in 23 cases with del(7q), 1 case with 7q CN-LOH, and 23 randomly selected cases identified a MLL3 truncating mutation leading to a premature stop codon in a case that lacked a 7q alteration. The del(11p13) contained only WT1 and primarily affected inv(16)-cases (5 of 7). Sequence analysis of WT1 in four cases with del(11p) revealed an additional frame shift mutation in the remaining allele in one case. Sequence analysis of WT1 in an additional 103 inv(16)-containing cases revealed mutations in 10 (9%) of the cases. The MDR at 17q11.2 was exclusively identified in inv(16)-containing cases (n=6) and included the tumor supressor NF1. Sequence analysis of all coding exons in NF1 in 4 additional inv(16)-containing AMLs revealed no additional mutation. Recurrent gains were identified at 22q11.21-q13.33 (n=20; 32 Mb), 8q24.21 (n=14; 138 Kb), 13q21.1-q34 (n=6; 14 Mb), and 11q25 (n=5; 368 Kb). The smallest gains were identified at 8q24.21 and 11q25, both containing only a single non-coding RNA gene (CCDC26 and LOC283177, respectively). Somatic CN-LOH were uncommon and only found at 1p36.33-p12 (n=2), 4q (n=2), and 19p (n=2). In a comparative analysis of paired diagnostic and relapse samples, novel CNAs at the time of relapse were identified at 3q13.31 (n=5), 5q (n=2), 17p (n=2), and 17q (n=2). The MDR at 3q was only 46 kb in size and contains a single transcript that has been connected to LSAMP, a putative tumor suppressor located 404 Kb upstream of the deletion. In summary, our data provide a comprehensive profiling of copy number alterations in pediatric and adult CBF AML. These data demonstrate a very low number of CNAs, with no significant differences noted between pediatric and adult cases. Interestingly, a number of novel recurrent secondary genetic alterations are identified. Exploring the biological role of these lesions in leukemogenesis and drug resistance should provide important insights into the CBF leukemias. Disclosures: No relevant conflicts of interest to declare.