Another powerful method used for identifying the location of chromosomal gains, losses, deletions, or amplifications, without prior knowledge of the chromosomal target that may be altered, is comparative genomic hybridization (CGH) (see Fig. 1). Briefly, isolated DNA from a patient’s marrow or tumor tissue is labeled with a one-color fluorochrome (e.g., red), whereas DNA isolated from normal control tissue is labeled with a different color (e.g., green). These differently labeled DNAs are hybridized against each other in a competitive hybridization reaction onto normal metaphase spreads. Computer-assisted image analysis detects colors generated after hybridization, which indicate equal hybridization, relative excess, or deficiency of the target DNA (relative to control). The ratio of color intensity provides a “copy number” karyotype.
Fig1. COMPLEMENTARY METHODOLOGIES OF IDENTIFYING VARIOUS GENOMIC ALTERATIONS AND THEIR RESOLUTIONS. See text for detailed descriptions of how these methodologies are applied for detection of genomic abnormalities in hematologic malignancies. Conventional cytogenetic methods detect clonal numerical and structural chromosomal abnormalities on a single cell level, at the resolution of 5 to 7 Mb. Multicolor FISH method with 24 different colors is specifically useful to identify the origin of marker chromosomes, complex 3- or more-way translocations, origin of ring chromosomes and other chromosomal abnormalities present in a complex karyotype such as those in MDS, lymphoma, and multiple myeloma. As mentioned in the text, there are at least five different reasons to perform interphase FISH in non-dividing cells from specimens of patients with hematologic malignancies. Interphase FISH is specifically useful for initial screening of the most recurrent rearrangements associated with CML, AML, ALL, and multiple myeloma as well as for detection of minimal residual disease with a diagnostic abnormality originally determined by conventional cytogenetics. In the lab of the author interphase FISH for PML-RARA fusion for the diagnosis of APL is a stat test with results obtained within 4 hours. Array CGH+SNP is a molecular method for detection of small and cryptic DNA changes at the exon-level resolution to 1 Mb. SNP platform is particularly useful for detection of acquired copy-number neutral chro mosomal regions as well as detection of chromothripsis (see text). Next-generation sequencing (NGS) is the most powerful method for detection of acquired somatic mutation at the single nucleotide level. Although not yet used routinely in clinical laboratories, the application of NGS to hematologic malignancies has revolutionized the current knowledge of many leukemic entities. aCGH, array comparative genomic hybridization; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; CML, chronic myeloid leukemia; FISH, fluorescence in situ hybridization; MDS, myelodysplastic syndrome; SNP, single-nucleotide polymorphism.
A particularly useful investigational approach is a “microchip array” in which labeled DNA or RNA from the sample of interest is hybridized with defined target sequences immobilized on a solid support system. This method can screen for genes that are gained/ amplified or deleted from the genome on a large scale or, in the case of RNA, to learn whether such genes are expressed at a particular stage of disease. The first example of successful application of the RNA microchip array technique was the discrimination of AML from acute lymphoblastic leukemia (ALL) based solely on gene expression. As shown in Fig. 1, in the array CGH (aCGH) procedure, large insert genomic clones, oligonucleotides, or single-nucleotide poly morphisms (SNPs) have replaced metaphase chromosomes used in routine CGH. Array CGH is a higher-resolution CGH technology of approximately 5 to 50 kb and provides diagnostic information for diseases associated with DNA dosage. It can also be used to discover previously unexpected sites of altered gene dosage associated with specific hematologic malignancies. The concept of obtaining gene copy number from multiple genome locations in a single measurement has been used to characterize numerous hematologic malignancies over the last 17 years, and its clinical utility is demonstrated throughout this chapter. Nowadays, SNP arrays are also used to genotype a few hundred to millions of SNPs in order to detect rare and common genomic rearrangements (see Fig. 1). These arrays require hybridization of only the test sample onto the array, unlike aCGH, which relies on co-hybridization of test and reference DNA. At this time aCGH+SNP arrays are used together in one study and they provide exon-level resolution of genomic changes.
The most advanced genomic technologies currently known is next generation sequencing (Chapter 3) or NGS (see Fig.1). These approaches use a range of techniques that enable sequencing of hundreds of thousands of nucleic acids simultaneously. In order of complexity, these approaches include sequencing of gene panels, exome sequencing (protein-coding genes), transcriptome (expressed RNA), and sequencing the whole-genome sequencing (WGS). Implementation of these techniques requires several arrays of several hundred thousand sequencing templates in parallel generating up to several hundred million short reads of DNA sequence per lane. The basic principle of NGS involves a process of DNA fragmentation, adapter ligation, and immobilization of the fragments via the adapters to create libraries. The libraries then undergo a process of amplification, generating multiple copies of each DNA fragment which are then sequenced in parallel by a fluorescence- or chemiluminescence based method, yielding billions of short sequence reads (Chapter 3). These reads are then aligned to the human reference genome and highly efficient algorithms are used to map these complex genomes. Because of its cost effectiveness and enormous sequencing capacity, NGS is revolutionizing hematologic malignancy research by facilitating the discovery of disease-initiating mutations, identifying novel drug targets, and allowing for the first time personalized treatment strategies for hematologic malignancies. Exome sequencing is a relatively inexpensive approach to identify protein-coding mutations, but is incapable of identifying structural genetic rearrangements, deletions, and insertions of DNA, a hallmark of many hematologic malignancies. Exome sequencing is performed at a depth of 100- to 200-fold coverage of the haploid genome, which enables detection of mutations present in leukemic subclones, which is important in the study of relapse. Transcriptome sequencing involves sequencing of genes actively expressed and can be adjusted to selectively study RNA transcripts that encode proteins (mRNA), transcripts regardless of coding potential (RNA), or a variety of small and noncoding RNA transcripts. RNA sequencing is a highly informative approach which enables identification of chromosomal rearrangements that result in the expression of chimeric fusion genes and sequence mutation detection. WGS usually involves sequencing leukemic and nonleukemic DNA from an individual. Although it is the most comprehensive modality, WGS may not identify all genetic alterations caused by variations in sequence coverage and difficulties in sequencing com plex and GC-rich regions of the genome (including gene promoters). These modern cytogenomic methods have increased the resolution at which chromosomal and gene rearrangements can be identified. Conventional cytogenetics, FISH, and aCGH+SNP along with NGS are complementary. Each has its own pros and cons in investigating genomic rearrangements of malignant cells.
Although conventional cytogenetics is the comprehensive study of all chromosomes, it requires a large number of dividing cells, which, in some diseases, such as myelofibrosis, is difficult to obtain (see Table 1). Furthermore, many small deletions or structural rearrangements are beyond the microscopic level of detection. FISH should be used in conjunction with conventional cytogenetics with both interphase and metaphase cells. It is a more sensitive method and detects rearrangements smaller than 1 kb. The main disadvantage of interphase FISH is that it cannot be used unless a known abnormality is suspected. When the abnormality is known, inter phase FISH identifies the clonal aberration at the single-cell level. aCGH+SNP provides genome-wide higher resolution analysis and important information concerning genomic changes in patients with a normal karyotype as well as acquired loss of heterozygosity, and chromothripsis (chromosome shattering), which are important prognostic and predictive tools. With the introduction of high throughput genomic technologies such as NGS, FISH-based chromosome level detection has gradually changed focus to genome-wide detection of single nucleotide and copy number variants that are common in leukemia. It is evident that identification of chromosomal aberrations by molecular cytogenomic techniques is important in detecting novel chromosomal rearrangements and genes involved in leukemogenesis. Understanding the basis of these techniques and their application is critical in the accurate diagnosis of hematologic malignancies. Table 1 describes the optimal algorithm and evolving new testing strategies. New technologies, capable of simultaneously detecting copy number changes, structural variants, and mutations, are expected to be used in future in a diagnostic setting.
Table1. Recommended Testing for Different Hematologic Malignancies
Genome sequencing has rapidly become routine practice. However, the intimate relationship between DNA sequencing, chromosome structure, and the position of chromosomes in the nucleus is not fully understood. The term “chromosomics” was recently proposed in order to integrate the original definition of cytogenetics (chromosomes and cytology) with genomics (gene content, structure and function) to ensure a closer integration of these fields. This term chromosomics is encompassing the integration of the latest advances in cytogenet ics, genome sequencing, epigenomics, and cell biology. Chromosomics approaches in future will lead to additional success in answering fundamental biological questions in hematologic malignancies.
