PCR for IG and TCR Gene Rearrangements

Leukemia-specific IG and TCR gene rearrangements identified at diagnosis can be used for MRD assessment of bone marrow (BM) or peripheral blood (PB) samples during and following treatment. However, similar IG and TCR gene rearrangements from normal lymphocytes in these specimens also are amplified. To discriminate between the leukemia-derived PCR products and PCR products of normal cells with comparable rearrangements, the amplified bands are subjected either to fingerprint66-68 or homo-heteroduplex analysis.69 Fingerprint analysis

Roche LightCycler

1G-1

Standard dot-blot semiquantitative analysis

Applied Biosystems ABI 5700

1G-5

V//Al/Tp-

/ / /

Jr

1B14 1 5161 7181920 2122 2B242526272829B0B1B2BBB4B5B6B7B8B94041424B44454647484950 Cycle Number

Roche LightCycler

0 5 10 15 20 25 30 35 40 45 50 Cycle Number

Applied Biosystems ABI 7700 Amplification-Pt#99 mrd probe Jh 1.2.4.5_

1GA 1

1GA G 1GA-1 1GA-2

1B14 1 5161 7181920 2122 2B242526272829B0B1B2BBB4B5B6B7B8B94041424B44454647484950 Cycle Number

0 5 10 15 20 25 30 35 40 45 50 Cycle Number

Applied Biosystems ABI 7700 Amplification-Pt#99 mrd probe Jh 1.2.4.5_

1GA-B

1G-

T—i—i—i i i—i—i i i—i—i—i—i—i—i—r

Figure 31-4. Assessment of the sensitivity of different methods for patient-specific IGH gene rearrangement assays.

consists of PCR amplification with a fluorescent primer and analysis by capillary electrophoresis, where clonal amplification results in a single peak within a background of polyclonal, constitutional amplification products.66-68 The homo-heteroduplex analysis takes advantage of the different migration properties in a polyacrylamide gel of V-J rearrangements containing a few mismatches (het-eroduplex) compared with fully matched V-J junctions (homoduplex).69 It has been recently reported that the use of a semiautomated electrophoretic technique (Phast-System) gives comparable results to traditional analysis for detection of clonality but is faster, simpler, and highly reproducible.70

Several studies have shown multiple IG and TCR gene rearrangements at diagnosis in childhood B-precursor ALL. Oligoclonal IGH rearrangements have been observed in 30% to 40% of cases and IGLK rearrangements in 5% to 10%, and are due to VH replacements, VH-DJH junction, or de novo IGH gene rearrangements.7174 The presence of oligoclonality is supposed to be a rare event in TCRB and TCRG gene rearrangements in B-lineage ALL.75-78 One exception was reported by Szczenpanski et al.,20 who observed TCRG oligoclonality in 38% of patients with B-lineage ALL. Oligoclonality of TCRD frequently are observed in incomplete rearrangements, VS2DS3 and Dd2Dd3.12,25,78 In T-lineage ALL, oligoclonality is rarely seen at diagnosis.75,76,79

Clonal evolution, that is, a difference from the original clone at relapse, has been detected in rearrangements of both the IG and TCR genes. Rearrangements of the IG genes are particularly sensitive to clonal evolution that occurs in more than 30% to 40% of cases. Clonal evolution of rearranged TCR genes is less frequent, being observed in 10% to 25% of cases. The risk of change in gene rearrangements seems to increase with time,but further change is not commonly observed in the second or third relapses.20,80-82 This clonal evolution may be due to the emergence of a new independent clone, to secondary leukemia, or, more commonly, to the subclones formed by continuous and secondary rearrangements of these genes.83-86

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