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.