title: Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.
abstract: The development of rapid polymerase chain reaction (PCR) protocols for amplification of rearranged heavy chain immunoglobulin (IgH) gene sequences has facilitated the identification of clonal IgH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias of B lineage. In the present report we have explored the recently described improved strategy for assessment of clonality of rearranged immunoglobulin heavy chain (IgH) genes in more detail in a series of 101 B cell malignancies and 50 polyclonal controls. The assay is based on an IgH-PCR with an automated fluorescence-based strategy for PCR detection of IgH gene rearrangements. Third complementarity determining region (IgH-CDR3) sequences were amplified using fluorescent dye labeled consensus primers homologous to the corresponding variable (V[H]) and joining (J[H]) gene segments in combination with a thermostable proofreading DNA polymerase. PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. PCR findings obtained with the optimized IgH-CDR3-PCR assay showed an overall monoclonality detection rate of 97% (97 of 101 cases with B cell neoplasms). The specificity was 100% as determined by analysis of 50 controls, all of which gave polyclonal PCR results. We found a high rate of monoclonal IgH-CDR3-PCR results not only in the leukemias and diffuse lymphoma but also in the group of follicular lymphoma, where a high rate of false negative results is frequently reported in the literature. In summary, we identified monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) with B cell lymphoma and in 42 of 42 (100%) cases with leukemia, immunocytoma and multiple myeloma. The results demonstrate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal lymphoid B cells. In combination with allele-specific primers the procedure may improve current experimental approaches to detect occult malginant B cells during initial staging and follow-up of NHL and ALL patients.
authors.affiliation: Department of Internal Medicine, Georg-August University, Goettingen, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Linke
authors.forename: I
authors.initials: I
authors.lastname: Bolz
authors.forename: A
authors.initials: A
authors.lastname: Fayyazi
authors.forename: M
authors.initials: M
authors.lastname: von Hofen
authors.forename: C
authors.initials: C
authors.lastname: Pott
authors.forename: J
authors.initials: J
authors.lastname: Bertram
authors.forename: W
authors.initials: W
authors.lastname: Hiddemann
authors.forename: M
authors.initials: M
authors.lastname: Kneba
mesh_terms: D000483:Alleles; D001483:Base Sequence; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007938:Leukemia / Q000235:genetics; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
chemical_list: D007143:Immunoglobulin Heavy Chains