Limitations and Difficulties of Comparative Genomic Hybridization

CGH only detects genomic aberrations that involve loss or gains of DNA sequences. Balanced translocations or inversions are therefore not detectable, nor are small intragenic rearrangements and point mutations. CGH also only detects DNA sequence copy number changes relative to the average copy number in the entire tumor sample. The relative green-to-red ratios can be transformed to indicate actual copy numbers if the absolute copy number in several loci are independently determined or if the ploidy is determined by DNA content analysis.6-54

Pericentromeric and heterochromatic repeat regions, unfortunately, cannot be reliably evaluated by CGH because unlabeled Cot-1 DNA blocks binding of the labeled DNA to the pericentromeric and heterochromatic regions. These DNA sequences are highly polymorphic in copy number between individuals. Thus, ratio changes at or near these regions should be interpreted cautiously, especially when the test and reference DNA samples come from different individuals.54

CGH, unfortunately, cannot detect single-copy losses or gains unless the extent of the region in loss/gain is greater than about 10 Mb. Moreover, the CGH ratio may not be a quantitative measure of the number of copies lost or gained unless the involved region is much greater than 10 Mb in extent.

Similar to other methods based on extracted DNA, CGH requires that the tumor specimens be relatively free from

Lett: Losses Right: Gains

- FTC cell lines (FTC133, FTC236, FTC238)

FIGURE 36-8. Summary of chromosomal aberrations analyzed by comparative genomic hybridization (CGH) in five thyroid cancer cell lines: (1) FTC-133 was derived from a primary follicular thyroid cancer (FTC); (2) FTC-236 was derived from a lymph node metastasis of FTC from the same patient as in FTC-133; (3) FTC-238 was derived from a lung metastasis of FTC from the same patient as in FTC-133; (4) AR081-1 was derived from an anaplastic thyroid cancer (ATC); and (5) TPC-1 was derived from a papillary thyroid cancer (PTC).

TABLE 36-6. Chromosomal Abnormalities Found in Anaplastic Thyroid Carcinomas by Comparative Genomic Hybridization

Common Region of Chromosomal Abnormalities

Study (Year)

Type of Tissue and Number

GAINS

LOSSES

Hemmer et al (1999)10 Komolke et al (1999)48

Wilkens et al (2000)49

Miura et al (2003)8

13 fresh-frozen or paraffin-erribedded tissues

8 cell lines

9 fresh-frozen tissues and 2 cell lines

10 fresh-frozen tissues and one cell line

5p (45%), 8q (37%), 3q (27%), 7p (27%). llq (27%), 20q (27%) lq (27%)

18q (88%), 16p (63%), 4p (50%), 8p (38%), 7p (38%), 7q (38%), 16q(38%) 9p (27%)

surrounding normal tissues that dilute the green-to-red ratio changes. If the normal tissue contribution is greater than 50% of the total DNA content, reliable detection of the ratio becomes increasingly difficult. In addition to normal cell contamination, intratumor genetic heterogeneity may also dilute the green-to-red ratio changes detected by CGH. This technique detects the average copy number of sequences in all cells included in the specimen, so those aberrations that are homogenously present (clonal) in the tumor cells are more readily detected. In most cases, this is an advantage, because the clonal changes are likely to represent the early and most important changes. However, in multiclonal tumors, the different genetic aberrations present in the individual clones may sometimes balance one another or exist at too low a frequency to be detected.54

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