PCR Detection of Antigen Receptor Gene Rearrangements

Although most follicular lymphomas exhibit t(14;18) and other lymphomas have characteristic chromosomal translocations, for patients with follicular lymphoma who do have a PCR amplifiable t(14;18), alternative strategies must be developed to detect MRD. Follicular lymphomas rearrange their Ig genes, providing a clonal marker that also can be amplified by PCR. The most

Follicular Lymphoma

Fig. 1. The bcl-2/IgH rearrangement results from the translocation of the bcl-2 proto-oncogene from chromosome 18 to the IgH locus on chromosome 14. Most breaks occur within the MBR within the 3' untranslated region. An mcr occurs some 15 kb downstream. PCR amplification of the derivative chromosome can be performed using primers 5' to the breakpoint and consensus JH primers.

Fig. 1. The bcl-2/IgH rearrangement results from the translocation of the bcl-2 proto-oncogene from chromosome 18 to the IgH locus on chromosome 14. Most breaks occur within the MBR within the 3' untranslated region. An mcr occurs some 15 kb downstream. PCR amplification of the derivative chromosome can be performed using primers 5' to the breakpoint and consensus JH primers.

hypervariable region of the Ig molecule is the third complementarity-determining region (CDRIII). The CDRIII is generated by the rearrangement of germline variable (V), diversity (D), and joining (J) region elements (24-26). The enzyme terminal deoxynucleotidyl transferase (TdT) inserts random nucle-otides at both the V-D and D-J junctions, and further diversity is generated by random excision of nucleotides by exonucleases (27). The final V-N-D-N-J sequence that comprises the CDRIII region is unique to that cell and acts as a unique marker for that leukemic clone. PCR amplification of the CDR III region is possible due to the presence of highly conserved sequences within the Variable (VH) and Joining (JH) regions. The large number of V regions makes the design of single oligonucleotide primer pairs capable of amplifying all Ig rearrangements difficult. At the time of initial presentation, deoxyribo-nucleic acid (DNA) from patients with lymphoma can be PCR amplified using consensus VH and JH region primers. Although these techniques have the advantage of being applicable to a greater number of patients, they are less sensitive than the detection of chromosomal translocations. More highly sensitive tumor detection can be achieved using primers directed against the unique junctional region sequences within the rearranged antigen receptor genes (28). The clonal product can then be directly sequenced and patient-specific probes constructed using N region nucleotide sequences. This strategy successfully amplifies and allows sequencing of the CDRIII to design patient allele-specific oligonucleotide to be used as oligonucleotide probes for the subsequent detec-

Antigen Receptor Gene Rearrangement

Fig. 2. PCR amplification of the complementarity determining region (CDR) III region of the IgH locus. PCR amplification can be performed using consensus primers to the FR3, resulting in a PCR product of 100-120 bp, or by using a series of family specific primers to the FR1, resulting in a PCR product of 300-350 bp.

Fig. 2. PCR amplification of the complementarity determining region (CDR) III region of the IgH locus. PCR amplification can be performed using consensus primers to the FR3, resulting in a PCR product of 100-120 bp, or by using a series of family specific primers to the FR1, resulting in a PCR product of 300-350 bp.

tion of MRD or as primers for second round or nested PCR amplification (2932). The strategies used to PCR amplify the CDRIII region is shown in Fig. 2 and use a variety of consensus primers from the framework regions (FR) of the V and J regions. The utility of PCR amplification of the CDR III region in B-cell malignancies, including anaplastic lymphocytic leukemia, myeloma, and chronic lymphocytic leukemia, has been demonstrated by many studies (3236). However, fewer studies have examined the clinical significance of MRD detection using Ig rearrangements in non-Hodgkin's lymphoma (32,36). Analysis of the IgH V genes in follicular lymphoma cells demonstrates that they have acquired somatic hypermutations and frequently demonstrate clonal variation, presumably resulting from ongoing activity of the mechanism of somatic hypermutation (37), making the use of consensus region primers more difficult.

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