Detection of Chromosomal Aberrations by Molecular ScreeningFISH

Although karyotype assessment provides a valuable framework for design of risk-adapted treatment of AML, it has some limitations as a means of determining the most suitable treatment approach for individual patients. Indeed, information derived from karyotype assessment may be substantially embellished by the results of molecular analy-

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Figure 30-4. Variation in cytogenetic risk group according to age. Cytogenetic risk group (see Table 30-6) was determined in 2677 patients derived from Medical Research Council AML10 and AML11 trials. Balanced chromosomal rearrangements associated with favorable risk were relatively more frequent in children and younger adults,whereas adverse karyotypic features were more common in older patients. (Reprinted from Grimwade D, Walker H, Harrison G,et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood. 2001;98:1312-1320. Copyright American Society of Hematology, used with permission.)

ses, which serve to further refine the diagnosis of AML, reliably identify subgroups of patients that require a specific treatment approach (e.g., molecularly targeted therapies), and define targets for subsequent MRD assessment.

A critical failing of cytogenetic analysis is that it may yield misleading results due to sampling of residual normal marrow elements and is unsuccessful in approximately 10% of patients, with rates differing according to whether samples are analyzed in local or more remote laboratories, entailing substantial differences in transit time. In such cases, RT-PCR, FISH, or both readily detect the presence of fusion genes corresponding to the favorable cytogenetic risk group. For patients lacking molecular evidence of fusion genes associated with favorable risk, interphase FISH may prove helpful in identifying lesions that would assign cases to the adverse risk group. These include evidence of loss of chromosome 5 or 7 material, a complex pattern of losses and gains, or presence of the BCR-ABL fusion. In non-APL patients, screening for involvement of MLL in the pathogenesis of AML can effectively be performed by Southern blot analysis. Although this is more labor-intensive than RT-PCR or FISH, it provides additional information indicating the presence of MLL cleavage or duplication events, as well as occurrence of translocations. It is not clear that screening for MLL abnormalities currently has any significant bearing on management of AML, but it may be useful in identifying additional patients with MLL-associated fusion genes who could be monitored for MRD by PCR-based approaches.

For those cases of AML with evaluable metaphases at diagnosis, patients may not necessarily be assigned to the most appropriate risk group on the basis of karyotype assessment alone, due to the occurrence of cryptic rearrangements. Studies conducted by the European Working Party and UK Medical Research Council (MRC) have established that almost 10% of cases with morphologic APL and successful karyotype analysis actually lack the classical t(15;17) but nevertheless have an underlying PML-RARA fusion.1619 This is most commonly the result of insertion events, in which chromosomes 15 and 17 are typically of normal appearance. Such cases share the beneficial response to targeted therapies, i.e., ATRA and arsenic tri-oxide. This is supported by outcome data from the UK MRC ATRA trial, which showed that patients in whom PML-RARA was identified solely by molecular means had a comparable outcome to those with t(15;17) documented by karyotype.19 This would suggest that cases of AML with cryptic rearrangement of CBF genes (i.e., with AML1-ETO or CBFB-MYH11) also are likely to be biologically equivalent to and share the favorable prognosis of those with karyotype-documented t(8;21) or inv(16)/t(16;16).

This supports the adoption of more widespread molecular screening for CBF leukemias in AML, which is most efficiently undertaken by RT-PCR. Evidence to date suggests that at least 10% of cases of AML with CBF leukemia have a cryptic rearrangement and that such cases cannot be reliably identified on the basis of distinct morphological features (reviewed in Reference 17). Where evidence of CBF leukemia is revealed by RT-PCR screening in the absence of the typical cytogenetic lesion, the result should be independently confirmed using FISH to document fusion gene formation, for example, as a result of an insertion event (see Figure 30-5). FISH comfirmation serves to exclude the possibility that the result was spurious due to PCR contamination or was unlikely to be of clinical relevance, should the fusion gene have arisen in a minor clone.

The relative value of molecular screening is dependent on the age group of patients to be screened, with CBF leukemias representing a greater proportion of AML arising in children and younger adults than in the elderly, where they comprise less than 5% of cases. The relative

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Figure30-5. Characterization by FISH of AML with fusion gene formation due to cryptic rearrangements. Cryptic (8;21) rearrangement in an AML M2 case demonstrated by FISH with AML1 (green) and ETO (red) probe set (Vysis, Downers Grove, IL). Fusion signals are seen on the derivative 21, and ETO signals are diminished on the derivative 8. (Figure prepared by Marina Lafage-Pochitaloff, Institut Paoli Calmettes, Marseille. Reprinted from Grimwade D. The clinical significance of cytogenetic abnormalities in acute myeloid leukaemia. BaillieresBestPract Res Clin Haematol. 2001:14:510; copyright 2001, with permission from Elsevier.)

Figure30-5. Characterization by FISH of AML with fusion gene formation due to cryptic rearrangements. Cryptic (8;21) rearrangement in an AML M2 case demonstrated by FISH with AML1 (green) and ETO (red) probe set (Vysis, Downers Grove, IL). Fusion signals are seen on the derivative 21, and ETO signals are diminished on the derivative 8. (Figure prepared by Marina Lafage-Pochitaloff, Institut Paoli Calmettes, Marseille. Reprinted from Grimwade D. The clinical significance of cytogenetic abnormalities in acute myeloid leukaemia. BaillieresBestPract Res Clin Haematol. 2001:14:510; copyright 2001, with permission from Elsevier.)

benefit of molecular screening also will vary according to the success of cytogenetic assessment, being most rewarding where cytogenetics has a relatively high failure rate or when there are no facilities for central review of kary-otypes. Molecular screening for CBF leukemias appears to be worthwhile because it enables the identification of additional patients who can be subject to MRD assessment using a fusion gene marker, as well as identifying individuals who can potentially be spared BMT in first CR. While cryptic CBFB-MYH11 and AML1-ETO gene fusions have been observed in a wide range of FAB types, the PML-RARA fusion is very closely correlated with M3/M3v morphology. Therefore, routine molecular screening for the PML-RARA fusion among all newly diagnosed cases of AML is not considered worthwhile but should be restricted to infrequent cases with severe coagulopathy, an APL-like immunophenotype, or cases with cells suggestive of APL morphology even if occurring in a minor cell population.

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