Detection of Clonal Evolution in Hematopoietic Malignancies by Combining Comparative Genomic Hybridization and Single Nucleotide Polymorphism Arrays Luise Hartmann, 1 Christine F. Stephenson, 1 Stephanie R. Verkamp, 1 Krystal R. Johnson, 1 Bettina Burnworth, 1 Kelle Hammock, 1 Lisa Eidenschink Brodersen, 1 Monica E. de Baca, 1 Denise A. Wells, 1 Michael R. Loken, 1 and Barbara K. Zehentner 1* BACKGROUND: Array comparative genomic hybridiza- tion (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome- wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenet- ics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows addi- tional identification of acquired uniparental disomy, a copy neutral aberration with known potential to con- tribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. METHODS: This study comprised 16 samples (acute my- eloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reas- signment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with re- sults obtained from conventional cytogenetic and FISH studies. RESULTS: Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log 2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample’s clonal composi- tion and genetic heterogeneity in 10 of 13 (77%) pa- tients. Moreover, in 3 patients, clonal disease progres- sion was revealed by array analysis that was not evident by cytogenetic or FISH studies. CONCLUSIONS: Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment tech- nique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry Array comparative genomic hybridization (aCGH) 2 has improved the analysis and characterization of he- matopoietic malignancies (1–4) by both discovering previously unknown copy number (CN) changes [of- ten small cryptic lesions that are not identified by con- ventional cytogenetic analysis or standard fluorescence in situ hybridization (FISH) testing] and comprehen- sively characterizing breakpoints of known aberrations (5–6). In plasma cell neoplasms (PCNs), a genetically heterogeneous disease group with inherently low pro- liferation rate, it has been shown that aCGH is superior for disease risk stratification compared with conven- tional cytogenetic or FISH studies (7). Similarly, aCGH allows better characterization of the 13q14 de- letion common in chronic lymphocytic leukemia (CLL). Deletions of 13q14 can be divided into 2 groups: low risk, which are lesions 2 Mb [tumor suppressor gene RB1 (retinoblastoma 1) 3 not deleted] and high risk, which are deletions 2 Mb (8). In contrast to aCGH, FISH, and conventional cy- togenetic studies, single nucleotide polymorphism 1 HematoLogics Inc., Seattle, WA. * Address correspondence to this author at: HematoLogics Inc., 3161 Elliott Ave, Suite 200, Seattle, WA 98121. Fax 206-223-5550; e-mail barbara@hematologics.com. Received May 29, 2014; accepted September 12, 2014. Previously published online at DOI: 10.1373/clinchem.2014.227785 © 2014 American Association for Clinical Chemistry 2 Nonstandard abbreviations: aCGH, array comparative genomic hybridization; CN, copy number; FISH, fluorescence in situ hybridization; PCN, plasma cell neoplasm; CLL, chronic lymphocytic leukemia; SNP, single nucleotide polymor- phism; UPD, uniparental disomy; MDS, myelodysplastic syndrome; AML, acute myeloid leukemia; DLRS, derivative log ratio spread; ASCN, allele-specific copy number; IL, interleukin; MLL, mixed-lineage leukemia; CBFB, core-binding fac- tor,  subunit; LOH, loss of heterozygosity. 3 Human genes: RB1, retinoblastoma 1; TP53, tumor protein p53; RUNX1T1, runt-related transcription factor 1, translocated to, 1 (cyclin D-related) (formerly ETO); RUNX1, runt-related transcription factor 1; OLIG2, oligodendrite lineage transcription factor 2; ERG, v-ets avian erythroblastosis virus E26 oncogene homolog; TMPRSS2, transmembrane protease, serine 2; U2AF1, U2 small nuclear RNA auxiliary factor 1; BCL2, B-cell CLL/lymphoma 2. Clinical Chemistry 60:12 1558–1568 (2014) Molecular Diagnostics and Genetics 1558 Downloaded from https://academic.oup.com/clinchem/article/60/12/1558/5621708 by guest on 17 October 2022