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Volume: Barrett's Esophagus
Chapter: Diagnosis
 

What are the newest techniques for evaluation of DNA rearrangements?

B.A. Jacobson, M. Araghi-Niknam, L.J. Ferrin (Minneapolis)

Reasons to study DNA rearrangements in cancer cells

Gross genome instability is a hallmark of malignancy, and modern molecular biology has provided numerous insights into how that instability contributes to the phenotype of malignancy. Thousands of recurrent genomic aberrations have been characterized and compiled [1]. At present, a guiding hypothesis is that an initial event causes instability of the genome of a somatic cell. The initial or subsequent events may occur either at random locations or at hot spots of instability, and multiple events are necessary on the pathway to malignancy. Mutations and transcriptional silencing contribute, but this paper will be concerned with gross rearrangements of large segments of DNA. There are many phenotypic characteristics that differentiate normal from malignant cells. Consequently, many rearrangements are almost always seen when the genome of a malignant clone is examined closely. Typically, there will be amplifications of one or two oncogenes, and deletions of a much larger number of tumor suppressor genes. Cells with very large rearrangements that affect the functions of essential genes will not be propagated, and consequently, examination of frequently rearranged regions often discloses genes important in tumorigenesis. Studies of these regions may also help delineate the underlying mechanism of the instability.

Technical overview

Many different methods are available for mapping gene rearrangements, and many currently used have been available for decades. The introduction of polymerase chain reaction (PCR) made the most impact in the field, and in the last decade, techniques have improved at an accelerating rate. Lately, improvements have been due to advances in computers, digital imaging, and the almost complete knowledge of the sequence of the human genome and the genes it encodes.

Considering all the methods, there is a trade-off between the cost of a method and the amount of information it provides, and the proper choice is guided by the goal of the experiment. It is becoming increasingly popular to use two complementary methods on the same tumor samples [2].

Candidate gene approaches

Often, an investigator wishes to know the status of one or a small number of genes in a specific tumor type. Given the almost complete human genome sequence, it is now fairly straightforward to clone any region from tumor DNA up to several kilobases in size by PCR, and screening for rearrangements can be done by Southern blotting. The advantage of this approach is that only general molecular biology expertise and equipment is necessary, but the disadvantage is that it is fairly labor intensive and this limits the size of the genomic region that can be sampled. An advance has come from the use of RecA protein to selectively cut and then clone desired fragments to isolate specific regions several hundred kilobases in size [3], but this technique requires extensive technical expertise.

Subtraction or comparison techniques

This set of technique exploits the differences between rearranged tumor DNA and matched normal DNA. The best known of these is representational difference analysis [4]. Fragments from normal DNA are capped with a unique oligonucleotide, denatured, and hybridized to a vast excess of tumor DNA. PCR of the mixture under conditions that require the unique caps on both strands of a renatured duplex then preferentially amplifies fragments of the normal genome that have been deleted in the tumor. A reversal of the DNA source can also be used to isolate amplified regions. This procedure produces clones, which can be sequenced, and the identity of the region determined by comparison to the human genome sequence.

In restriction landmark genomic scanning, DNA is digested by a restriction enzyme and run on an agarose gel. The lane is removed, digested in situ using a different enzyme, and laid across the origin of a second gel. This two-dimensional technique allows the resolution of thousands of fragments of DNA. The image from tumor DNA is compared to that of normal DNA, and spots that differ in intensity or position can be excised and cloned. A conceptually similar technique called inter-(simple sequence repeat) PCR uses a series of repetitive primers to amplify a subset of the genome. The products from tumor and normal DNA are then analyzed on gels. Several hundred bands can be analyzed in this way and represent a fairly unbiased sampling of the genome [5].

The advantages are that they require only inexpensive equipment and they produce clones that can be sequenced to uniquely identify their positions in the human genome. However, they are labor intensive and may miss a significant number of rearrangements.

Karyotyping

While a standard karyotype analysis can identify the chromosomal location of a rearrangement, many tumor genomes are too complicated to be analyzed unambiguously, and fluorescence in situ hybridization and spectral karyotyping have become popular. With these techniques, a fluorescently labeled DNA probe is hybridized to a metaphase chromosome spread from the tumor, and its position observed in relation to standard chromosome bands. In spectral karyotyping, a set of probes from an entire chromosome can be used to highlight any rearranged material derived from that chromosome. Probes that paint each chromosome are available and a variety of different fluors can be used [6].

In comparative genomic hybridization, tumor DNA is labeled with a fluor, and normal DNA is labeled with a second fluor. They are mixed and hybridized to a normal metaphase chromosome spread. The image is processed digitally and the amount of tumor and normal DNA at each chromosomal position determined. The normalized amount of tumor signal gives an estimate of the copy number. The method is especially sensitive for detecting tumor DNA amplifications, which usually contain numerous copies of a region.

The advantages are that the entire genome is examined and the chromosomal position of the rearrangement is determined. The major disadvantages are the equipment cost and the technical expertise. Generally, a rearrangement can only be positioned with a precision of about ten megabases (the size of a visible chromosome band), but this information can be used as a springboard for other methods that have a finer resolution.

Allelotyping

This strategy is often used to map deletions by detecting loss of heterozygosity. There are thousands of known positions where most people have inherited two alleles of different size or sequence, and markers from these regions can be amplified by PCR and scored by a variety of techniques, usually on a polyacrylamide gel. If tumor DNA shows a loss of an allele, there has been a deletion in that region. The newest variation involves the use of single nucleotide polymorphisms, where hundreds of thousands of markers are available [7]. The advantage is that a fairly precise positioning of a rearrangement can easily be made (limited by the number of PCR primers that can be purchased). Large scale mapping is tedious and requires the use of automated equipment or a commercial genotyping service such as Research Genetics, Huntsville, AL.

Microarrays

This technology, usually used in gene expression studies, is the newest and most powerful method available. It involves constructing a microarray of mapped clones on slides, hybridization of tumor and normal DNA to slides, and comparison of the respective spot intensities. The copy number of each clone in the sample DNAs can be determined simultaneously at thousands of positions [8]. For those interested in the status of a single gene in a large number of tumor samples, a microarray with 645 individual tumor specimens has been constructed [9].

Future directions

Because the mapping of genes, rearrangements, and the associated breakpoints has undergone massive technological improvement, this facet of cancer research will soon cease to be a limiting factor. Now there is an acute need for effective bioinformatics to process the massive amount of data being produced, and this will be especially evident once microarray data becomes readily available. Efforts will soon turn to how the higher order effects of genetic shuffling affect control of the cancerous cell. Another intriguing area is the mechanism behind the marked instability of tumor cells. The hope is that the instability can be turned against them, or at least be used to render them a stationary target.

References

1. Mitelman F, Mertens F, Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997;15:417-474.

2. Yu F, Jensen, RT, Lubensky IA, Mahlamaki EH, Zheng YL, Herr AM, Ferrin LJ. Survey of genetic alterations in gastrinomas. Cancer Res 2000;60:5536-5542.

3. Ferrin LJ. Flexible genetic engineering using RecA protein. Methods Mol Biol 2000;152:135-147.

4. Lisitsyn N, Wigler M. Representational difference analysis in detection of genetic lesions in cancer. Methods Enzymol 1995;254:291-304.

5. Stoler DL, Chen N, Basik M, Kahlenberg MS, Rodrigues-Bigas MA, Petrelli NJ, Anderson GR. The onset and extent of genomic instability in sporadic colorectal tumor progression. Proc Natl Acad Sci USA 1999;96:15121-15126.

6. Padilla-Nash HN, Nash WG, Padilla GM, Roberson KM, Robertson CN, Macville M, Schrock E, Ried T. Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping. Genes Chromosomes Cancer 1999;25:53-59.

7. Yang Z, Wong BK, Eberle MA, Kibukawa M, Passey DA, Hughes WR, Kruglyak L, Yu J. Sampling SNPs. Nat Genet 2000;26:13-14.

8. Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D, Brown PO. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet 1999;23:41-46.

9. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch M, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844847.


Publication date: August 2003 OESO©2015