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PCR Detection of Tumor Cells 185
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Ultrasensitive PCR Detection of Tumor Cells in Myeloma
Friedrich W. Cremer and Marion Moos
1. Introduction
Chromosomal aberrations, such as translocations or inversions, described for a
growing number of malignancies, are now widely used to detect tumor cells by
polymerase chain reaction (PCR). However, in multiple myeloma (MM), no such
ubiquitous PCR marker exists. Therefore, other means have been established to
distinguish myeloma cells from normal cells. Because the plasma cells of a myeloma
clone share an identical rearranged immunoglobulin gene sequence, it is possible to
detect malignant cells with PCR primers specific for the VDJ rearrangement of the
heavy chain of each myeloma clone. The sensitivity and specificity of this method,
named allele-specific oligonucleotide (ASO) PCR, even with low proportions of
malignant cells, has been proven (1).
The heavy chains of the immunoglobulins have three highly variable regions near
their amino-terminal end that mediate specific binding of the antigen. These regions
are called complementarity determining regions (CDR1 to –3). The sequences of the
heavy chain of the immunoglobulin genes are encoded on chromosome 14. In germline
configuration, several hundred base pairs divide the approx 200 variable regions
(V) from the 30 diversity regions (D). Several kilobase pairs downstream, 6 joining
regions (J) are located. About 7000 base pairs to the 3′-end the constant regions of the
heavy chain are encoded. During B-cell maturation, a random rearrangement of
these regions occurs, in which one of the V-, one of the D-, and one of the J-regions are
joined together, thus generating the VDJ-segment. Together with the sequence of the
rearranged light chain, it determines the antigenic specificity of the immunoglobulin.
The rearrangement of V-, D-, and J-regions can generate over 35,000 different VDJsequences.
The diversity of these sequences is further increased by three different
mechanisms: (1) the recombination process is imprecise, thus nucleotides can be lost
or stretches of bases that divide the different regions are not deleted; (2) nucleotides
can be inserted without matrix at the junctions of V-, D-, and J-regions; and (3) somatic
hypermutations, especially of the V-regions, further enhance the antigenic specificity of
the immunoglobulin. Although CDR1- and CDR2-regions are entirely encoded by the
V-region, the CDR3-region stretches over the 3′-end of the V-region, the D-region
and the 5′-end of the J-region (see Fig. 1). Thus, the CDR3-region has the highest
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
185