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nucleotide sequencing

nucleotide sequencing

has been demonstrated in detecting t(9;22) in CML and t(8;21) in AML (78). Thus, rather than throwing the baby out with the bath water, such studies could guide the use of FISH along with chromosomal banding. This strategy is especially useful for evolving diseases like CML or MDS. While tracing the original cytogenetically marked clone by FISH, the metaphase analysis in these diseases could provide additional insights into evolving chromosomal aberrations that might be of prognostic significance. In this respect, an additional M-FISH analysis would further help in identifying the origin of the marker chromosome.

Polymerase chain reaction amplification technique extends the sensitivity of FISH further. However, as shown in Table 6, several crucial differences in the principles of these two assays warrant expert discretion in employing these assays judiciously. FISH seems to be limited in the detection level of 50-100 kbp change, whereas PCR combined with sequencing could potentially detect even a single basepair change (48). The major factors that affect the sensitivity of FISH are the probe size, labeling scheme, size and orientation of the target cell nuclei, and the number of nuclei enumerated for quantitation. In a recent study, it was found that the gap between FISH and PCR resulting from a relatively large size of the hybridization probe used and the lack of amplification could, at least in some instances, be bridged by using additional FISH on initial negative samples employing smaller probes (81). In contrast, because PCR techniques rely heavily on the use of specific primers, often the assay could underestimate the regional rearrangement involving several breakpoints, which could easily be detected by FISH, as seen in a study detecting immunoglobulin light-chain mRNA in B-cell lymphoma (82). Similarly, the scattering of the 11q13 breakpoints during t(11;14) in mantle cell lymphoma resulted in severely low detectability of the translocation with genomic PCR as compared to interphase-FISH (83). Another problem often noted with PCR/RT-PCR is a high frequency of false-positive cases, such as those reported by GleiBner et al. (84) in adult acute B-lineage lymphoblastic leukemia. FISH is thus warranted in such cases to rule out false-positivity. Furthermore, it might be noted that sometimes genetic changes can be detectable prior to their transcription into detectable levels of abnormal mRNA. It is especially relevant in posttherapy disease monitoring for predicting relapse. Quantitative real-time RT-PCR has been considered to be the assay of choice for monitoring the post-bone-marrow-transplant cases of CML (75). However, a study conducted in 2002 on a large serial specimen set from 78 posttransplant CML patients showed that, in some cases with relapse, the increase in FISH positivity preceded that of the quantititative RT-PCR (85). The genomic changes could also precede the appearance of detectable levels of protein as, often, FISH-positive cases are not matched by immunohistochemistry; for example, in mantle cell lymphoma, although t(11;14) was detected by FISH in 97% cases, only 69% were simultaneously positive for cyclin D1 protein immunoreactivity (83). As mentioned earlier, FISH also offers the luxury of following the biology of an abnormal clone through the malignancy evolution and posttherapy regression. Recently, a detection of telomerase mRNA by in situ PCR in cyto-genetically abnormal clones localized simultaneously using various CEP probes demonstrated that a disappearance of telomerase-expressing abnormal clones in AML correlated with a response to chemotherapy (86). Further, the feasibility of cell-by-cell analysis with FISH is of special value. PCR/RT-PCR techniques that use isolated nucleic acids could show false-negative results if they have overpowering contamination from normal cells. The resolution to this problem is tissue microdissection, which at the moment is extremely tedious, demands special skills, and, hence, is less prevalent.

Clearly, FISH provides several unique advantages compared to the other molecular tools available. The choice of the molecular assay should rely on the clinical objective, ease of application, cost-effectiveness, nature of clinical specimen, and so forth. Also, the interpretation of data, especially in case of false-negative and false-positive cases, should be validated with another molecular technique. In the case of FISH, as also well demonstrated in cases of bcr-abl and PML-RARa, unbalanced deletions or insertion with or without duplication would result in variable labeling patterns that necessitate intelligible interpretation, as reported recently (37,87).

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