Abstract
Abstract 3550
Introduction:
The genetic loci altered in many de novo leukemia cases are relatively well-understood and can be accurately assessed by current cytogenetic techniques including multi-probe fluorescence in situ hybridization (FISH). However, identifying the cancer genes involved in complex leukemia karyotypes remains problematic due to the presence of multiple secondary structural rearrangements observed in subclonal populations. These alterations often affect both chromosome (chr) homologues and predominantly involve chr 1, 3, 5, 7, 12 and 17. Such clonal diversity within a tumor reflects the underlying biologically-selected sequential and multiple rearrangements and can, if carefully mapped, highlight the locations of tumor suppressor genes and modifiers involved in disease progression. Previous generations of DNA microarrays have proven useful in dissecting genomic changes in the predominant tumor clone, including copy-neutral loss of heterozygosity (CN-LOH) when single nucleotide polymorphism (SNP) arrays are used. However, a well-known shortcoming of DNA microarrays to date has been their limited sensitivity for accurately detecting low level mosaicism (<20%) and subclonal changes that are common in complex karyotypes.
Methods:
Using leukemia cases that showed complex karyotypes with up to 4 subclones, we compared the ability of standard (SNP 6.0, Affymetrix) and next-generation (Cytoscan HD, Affymetrix) SNP/copy number oligonucleotide arrays to accurately detect the observed karyotypic subclones and more precisely delineate areas of complex chromosomal alterations. Genomic DNA extracted from fresh material or 24∼48 hour short-term cultures from 8 patients with either de novo or previously treated chronic lymphocytic leukemia (CLL) was assessed on the SNP 6.0 and Cytoscan HD platforms and then compared with their karyotype, and/or supporting FISH studies. Copy number alterations and CN-LOH calls were made using ChAS software (Affymetrix), with the degree of clonal mosaicism analyzed for segmental increments of each chromosome by averaging the smooth signal data.
Results and Conclusion:
For all 53 CN-LOH and copy number calls, the two arrays gave identical detection rates and similar alteration boundaries in 34 instances (64.1% concordance). The genetic alterations that differed among the cytogenetically-related clones (subclones) were subclonal, in all but 3 instances, and most frequently involved chr 1 and 5. In general, the Cytoscan HD arrays were able to accurately detect karyotypically-confirmed subclones down to the 20% level (as well as distinguishing 90% vs. 100% calls), as opposed to the 30–50% level seen with the SNP 6.0 arrays. Improved detection of the discrete subclones or lower level clonality was attributed to more precise allele peak heights that did not require smoothing. Next-generation SNP/copy number oligonucleotide arrays show great promise in providing additive value to leukemic genomic profiling by clear visual separation of multiple genomic alterations within clonally diverse samples with the potential of identifying novel genetic alterations that may be important in disease progression.
Disclosures:
No relevant conflicts of interest to declare.
Single-Nucleotide Polymorphism Markers from De-Novo Assembly of the Pomegranate Transcriptome Reveal Germplasm Genetic Diversity
