Methods for assessing risk are based on the presence of clonal abnormalities in hematopoietic cells. These methods are shown in Table 5.2 and include standard cytogenetics, interphase FISH, analysis for LOH, PCR for point mutations, and X-inactivation-based clonality assays. Each of these approaches has strengths and weaknesses in this context.
Standard cytogenetics analyzes a limited number of cells that must be capable of mitosis and therefore lacks sensitivity and specificity. Most patients who develop t-MDS/AML after ASCT may have normal cytogenetics at the time of stem cell harvest, whereas some patients who have characteristic cytogenetic abnormalities will not develop t-MDS/AML.
Interphase FISH (Figure 5.2) circumvents some of the frailties of conventional cytogenetics. For example, abnormal clones (5q-, -7, +8, -11) were detectable in pre-ASCT specimens from nine out of 12 patients who developed t-MDS/ AML. An advantage of interphase FISH is that hundreds of non-mitotic cells can be analyzed. However, the technique is locus-specific and requires prior selection of markers for analysis, such as 5q-, 7q- and +8. In addition, interphase FISH is
Table 5.2 Methods for assessing the risk of t-MDS/AML before and after ASCT.
1. Standard cytogenetics
2. Interphase FISH
3. Loss of heterozygosity (LOH)
4. PCR for point mutations
5. X-inactivation-based clonality
Fig. 5.2 Applications of interphase FISH to detect trisomy, monosomy or chromosomal deletions o o o w
PCR of germline tissue
PCR of MDS cells
Fig. 5.3 Schematic representation of loss of heterozygosity (LOH) in MDS
In the presence of two alleles, two bands will be apparent after PCR amplification, whereas a single band will be seen if there is allelic deletion.
not sensitive below the level of approximately 5-10% of cells. However, the identification of clonal abnormalities in a high percentage of cells may indicate a proliferative advantage for these cells, and may be more predictive of the development of t-MDS/AML. The specificity of interphase FISH is also unknown, since we do not know how many patients who do not develop t-MDS/AML have interphase FISH abnormalities at the time of stem cell harvest. The test has been validated only in retrospective studies, and it is time- and labor-intensive as a screening test.
LOH analysis is based on the loss of one allele at a particular locus, usually by PCR analysis. This strategy can be used to identify LOH and to define the excursion of large deletions (Figures 5.3 and 5.4). It is a population-based assay and requires prior selection of loci to be analyzed. It lacks sensitivity and is probably unable to detect fewer than 20% of cells with LOH at a given locus. However, it is more likely to be specific, in that a positive test indicates clonal expansion of cells with LOH. It is amenable to high-throughput strategies, but has not yet been validated as a predictor of post-ASCT t-MDS/ AML in prospective studies, although such studies are under way.
PCR for point mutations or chromosomal translocations is emerging as a potentially useful predictor of t-MDS/AML as we learn more about the molecular genetics of the disease.
Marker 1 Marker 2 Marker 3
• The deletion is bordered by markers 1 and 3
Fig. 5.4 Schematic representation of the use of LOH to map deletions
Markers that may be useful include mutations in RAS, FLT3, AML1 and M LL. In addition, PCR can be used to identify fusion transcripts, including AML1/EVI1, PML/RARa and 11q23 gene rearrangements. The PCR approach is also by definition locus-specific, and there are relatively few markers known to date. It is highly sensitive and capable of detecting only a few cells. But, as noted above, since some normal individuals harbor PCR-detectable rearrangements, the specificity of the assay remains to be determined in this context. The test is probably most informative when performed using quantitative techniques, such as the Taqman PCR, and is amenable to high-throughput analysis, but has not yet been validated as a predictor of t-MDS/AML.
X-inactivation-based clonality assays require no locus-specific information, or indeed any information about the nature of the mutation that causes t-MDS/AML (Figure 5.5). It detects only clonal populations of cells that have a prolif-erative advantage over normal polyclonal cells. It uses DNA, is PCR-based and is readily amenable to high-throughput analysis, but is only applicable to female patients. There are several potential pitfalls of this test, including false-positive
Schema for the human androgen receptor assay (HUMARA)
1 Variable length CAG expansion repeat distinguishes the maternal from paternal X in >90% of females 1 Variably methylated Hpa II sites distinguish active from inactive X chromosomes
Fig. 5.5 Human androgen receptor assay (HUMARA)
(a) Schema of the assay, which uses the variable-length CAG repeat pattern to distinguish the maternal and paternal X chromosomes. (Continued.)
Results from the human androgen receptor (HUMARA) clonality assay
Fig. 5.5 Human androgen receptor assay (HUMARA) (continued)
(b) Two bands will be seen after PCR amplification in polyclonal cells where there is random inactivation.
(c) A single band will be seen in a clonal population.
(d) Results from patients studied, showing polyclonal, oligoclonal and clonal populations.
Cleaves active alleles
Cleaves active alleles
Results pattern Oligoclonal
tests due to germline or acquired skewing of the pattern of may be difficult to interpret in cases with severe skewing of the X-inactivation. This problem can be overcome in part by the X-inactivation pattern. This technique has been validated in appropriate use of related tissue controls. However, the test retrospective studies and prospective studies are ongoing.
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