PCR of Gene Rearrangements for the Detection of Minimal Residual Disease in Childhood ALL

互联网2014-02-13

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The study of submicroscopic or minimal residual disease (MRD) in childhood acute lymphoblastic leukemia may eventually lead to stratification of therapy on an individual patient basis (reviewed in ref. 1 ). PCR of immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements provides widely informative markers (Table 1 ), which, in the majority of cases, are stable during the disease course (2 ). Generation of leukemia-specific probes using this technique allows detection of MRD at levels of one leukemic cell in 10,000 to 100,000 normal bone marrow mononuclear cells (BM MNC).

Table 1 Primer Systems ^a

Locus	No^b	Primer sense (ref)	No^b	Primer antisense (ref)	[Mg²⁺ ]	Size, bp	+B	+T-
IgH FR3	1	5′-ACACGGC(C/T)(G/C)TGTATTACTGT-3′ (2)	3	5′-GTGACCAGGGT(C/T)C C(C/T)TGGCCCCAG-3′ (2)	1.5–30	65–155	75%	8%
			4	5′-AACTGCAGAGGAGACGGTGACC-3′ (3)	15–30	80–170
			2	5′-GACCAGGGT(C/T)C C(C/T)TGGCCCCAG-3′^e
Vδ2-Dδ3	5	5′-CTTGCACCATCAGAGAGAGA-3′ (2)	7	5′-GTTTTTGTACAGGTCTCTGT-3′	10–30	100–150	45%	4%
			8	5′-AGGGAAATGCACTTTTGCC-3′ (2)	15–30	110–170
			6	5′-TTTTGTACAGGTCTCTGT-3′ ^c
Vδ1-Jδ1		5′-GCCTTACAGCTAGAAGATTC-3′		5′-GTTCCTTTTCCAAAGATGAG-3′	15–30	80–150	5%	25%
VγI-Jγ1/2^d		5′-TG(A/C)(C/T)TCTGG(A/G)GTCTATTACTGT-3′		5′-CGATACTTACCTGTGACAAC(C/A)AG-3′	30	80–160	45%	90%^d
VγII-Jγ-1/2^d		5′-AAACAGGACATAGCTACCTACT-3′		5′-CGATACTTACCTGTGACAAACC/AAG-3′	30	80–160	45%	90%^d
Lead Vγ2-anti Vγ2		5′-GTCATGTCAGCCATTGAGTT-3′		5′-TCTCTCTCTGATGGTGCAAG-3′	15	220	control	control

^a The primer shown here use a common buffer and generate products that can easily be resolved on 8% PAGE ^b Refers to Fig. 1 ^c Sequencing primers ^d These primers can be used in a multiplex reaction. The VγI primer is a consensus primer and amplifies all members of this group except Vγ7 + B and +T indicate the percentage of patients by lineage expected to show a clonal rearangement at each locus at diagnosis.

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