title: Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity.
abstract: Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (r Tth DNA Polymerase, XL) providing both 5'-->3' polymerase and 3'-->5' exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The r Tth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the r Tth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.
authors.affiliation: Hematology Unit, 2nd Department of Internal Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Timea P
authors.initials: TP
authors.lastname: Gurbity
authors.forename: Eniko
authors.initials: E
authors.lastname: Bagdi
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Groen
authors.forename: Leo M
authors.initials: LM
authors.lastname: Budel
authors.forename: Mustaffa
authors.initials: M
authors.lastname: Abbou
authors.forename: Laszlo
authors.initials: L
authors.lastname: Krenacs
authors.forename: Winand N M
authors.initials: WN
authors.lastname: Dinjens
mesh_terms: D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D005557:Formaldehyde; D015322:Gene Rearrangement, B-Lymphocyte; D005803:Genes, Immunoglobulin; D006801:Humans; D016393:Lymphoma, B-Cell / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; D005557:Formaldehyde; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases