Copy Number Abnormalities

Gains (amplifications) or losses (deletions) of chromosomal mate rial at specific loci are recognized as playing an important role in the pathophysiology of cancer by either amplifying oncogene expression or decreasing tumor suppressor gene activity. In the germline, trisomy 21, for example, predisposes individuals to transient myeloproliferative disorders and acute megakaryoblastic leukemia.[1] Deletions at the RB1 locus encoding the retinoblastoma gene or deletions of the TP53 gene encoding the p53 tumor suppressor both predispose to the development of cancer.[2] In a landmark set of studies, it was shown that tumors from patients who inherit a mutant copy of the retinoblastoma tumor suppressor gene often have deletions of the remaining allele. This process has been termed loss of heterozygosity, and searching for such events in tumor samples has been used as a tool to identify genes involved in cancer progression. Similarly, amplification of genomic locus can play an important role in cancer progression.[3] For example, in multiple myeloma, amplification of a small amplicon at chromosome 1q23 is associated with adverse prognosis.[4]

Identifying CNAs has been done using a number of techniques. The original method was with metaphase cytogenetic analysis, which can identify abnormalities affecting large regions of the genome and was the basis for many important initial insights in hematologic malignancies. More recently, methods for assessing CNAs have advanced to include comparative genomic hybridization (CGH) and high-density single nucleotide polymorphism mapping arrays. However, these are being replaced by massively parallel genome sequencing. Although cytogenetic analysis remains a part of the diagnostic work-up for new cases of leukemia, it is likely that it will be replaced by NGS methods that also have the ability to detect point mutations, deletions/insertions, copy number changes, and chromosomal translocations, all at high resolution.

To identify statistically significant regions of CNAs, algorithms such as the genomic identification of significant targets in cancer (GISTIC) have been developed which can plot regions of amplification and deletion.[5]

Chromosomal Rearrangements

 Translocations were among the very first genomic defects to be discovered in cancer because cytogenetic analysis of metaphase chromosome spreads was feasible on cell lines, especially for the acute leukemias. Chromosomal rearrangements include balanced and unbalanced translocations, inversions, and complex aberrations. Two basic types of translocations are common: those that result in fusion proteins involving two distinct genes and those that result in overexpression of an otherwise structurally normal gene. Translocations resulting in fusion transcripts (e.g., ETV6/RUNX1 in ALL) generally involve chromosomal breakage within intronic regions of the two genes, with in-frame fusion producing a new protein with a novel function being a result of the normal process of RNA splicing.[6]

In contrast, translocations resulting in overexpression typically involve the juxtaposition of a coding region next to a highly active promoter or enhancer region, such as an immunoglobulin region in B cells. For example, in follicular lymphoma, translocations frequently involve juxtaposition of the antiapoptotic gene BCL2 to the immunoglobulin heavy chain enhancer region, leading to massive overexpression of BCL2 RNA and protein.[7]

Complex chained rearrangements termed chromoplexy and regions of massive chromosomal rearrangement termed chromothripsis are more frequent than previously thought (Fig. 1).[8]

For the discovery of novel translocations, either whole genome sequencing or RNA-seq are the optimum methods. However, when a distinct fusion characterizes a specific disease (e.g., chronic myeloid leukemia [CML] and the BCR/ABL fusion), specific PCR reactions to detect it can be used for both diagnosis and response following therapy.[9]

Epigenomics

Epigenetic gene regulatory mechanisms play a critical role in the regulation of transcription, DNA repair, and replication. Several large scale profiling efforts (e.g., through the National Institutes of Health ENCODE [Encyclopedia of DNA Elements] project) have used these technologies to annotate cancer cell lines and normal human and murine tissues, including hematopoietic subsets. Sequencing approaches to identify epigenomic changes include chromatin immunoprecipitation followed by sequencing (ChIP-seq), micrococcal nuclease (MNase) sequencing, DNAse sequencing (DNAse-seq), bisulfite sequencing and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), and a range of chromatin capture techniques including HiC (high-throughput chroma tin conformation capture). Massively parallel sequencing, coupled with bisulfite sequencing approaches, allows for genome-wide assessment of DNA methylation in development and disease.

Modifications to histones are orchestrated and tightly regulated by a group of enzymes called chromatin regulators. Perhaps one of the most striking results derived from genome-wide sequencing analyses in cancer is the frequency of somatic mutations in chromatin regulators, which account for up to 25% of all cancer drivers. With the use of NGS techniques combined with chromatin immunoprecipitation, it is now possible to comprehensively investigate the molecular mechanisms of epigenetic alterations and define their disease relevance. ChIP-seq can be used to map histone modifications that are associated with actively transcribed regions, repressed regions, or regions found at distal regulatory elements. Single-cell sequencing–NGS applications have been developed that allow DNA and RNA-seq of single cells derived from the tumor as well from the tumor microenvironment. A widely used approach takes advantage of packaging single cells into an emulsion droplet; when combined with a molecular bar coding of every RNA molecule from each single cell and then RNA seq of the entire population, it is possible to precisely assign each RNA molecule to each cell, making it possible to determine the gene expression profile of each single cell. This approach allows in-depth dissection of the tumor and its subclonal structure. Perhaps the major use of this approach will be to identify the nature of the cells of the microenvironment and how it is altered by infiltrating tumor cells.

Fig1. CARTOONS DEPICTING THE MAJOR COMPLEX STRUCTURAL VARIANTS. Chromothripsis, templated insertion, and chromoplexy.