Improved clonality detection in B‐cell lymphoma using a semi‐nested modification of the BIOMED‐2 PCR assay for IGH rearrangement: A paraffin‐embedded tissue study

Abstract

The BIOMED-2 PCR protocol for targeting the IGH gene is widely employed for detecting clonality in B-cell malignancies. Unfortunately, the detection of clonality with this method is not very sensitive when paraffin sections are used as a DNA source. To increase the sensitivity, we devised a semi-nested modification of a JH consensus primer. The clonality detection rates of three assays were compared: the standard BIOMED-2, BIOMED-2 assay followed by BIOMED-2 re-amplification, and BIOMED-2 assay followed by semi-nested BIOMED-2. We tested more than 100 cases using paraffin-embedded tissues of various B-cell lymphomas, and found that the clonality detection rates with the above three assays were 63.9%, 79.6%, and 88.0%, respectively. While BIOMED-2 re-amplification was significantly more sensitive than the standard BIOMED-2, the semi-nested BIOMED-2 was significantly more sensitive than both the standard BIOMED-2 and BIOMED-2 re-amplification. An increase in sensitivity was observed in all lymphoma subtypes examined. In conclusion, tumor clonality may be detected in nearly 90% of B-cell lymphoma cases with semi-nested BIOMED-2. This ancillary assay may be useful when the standard BIOMED-2 fails to detect clonality in histopathologically suspected B-cell lymphomas.

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses

“…At the present time, which has seen rapid strides in the development of omics analysis technologies, such as genome analysis, it is expected that data‐driven studies based on analysis of clinical samples will help clarify the molecular mechanisms responsible for the occurrence, progression and treatment responsiveness of diseases, thereby leading to the development of new biomarkers for diagnosis and identification of drug discovery targets . In particular, analysis of pathological tissue samples collected from the exact site of a disease, such as cancer, will be indispensable for the realization of genomic medicine …”

Improved clonality detection in Hodgkin lymphoma using a semi‐nested modification of the BIOMED‐2 PCR assay for IGH and IGK rearrangements: A paraffin‐embedded tissue study

“…We carried out semi‐nested modifications of the BIOMED‐2 protocols for IGH and IGK rearrangements. For the IGH clonality assay, we designed three primers located three bases internal to the JH consensus primer as described elsewhere (Table ) . For the IGK clonality assay, we designed four primers three bases internal to the Jk primers and one primer that was eight bases internal to the Kde primer (Table ).…”

“…In the preliminary experiment, two types of detection techniques were compared; heteroduplex electrophoresis in a 6% polyacrylamide gel and DNA‐chip‐based electrophoresis (Agilent Bioanalyzer 2100, Agilent Technologies, Santa Clara, CA, USA) . We found that the latter technique, which is rapid and simple, and can be performed at a low cost per sample, was more sensitive than the former.…”

“…We found that the latter technique, which is rapid and simple, and can be performed at a low cost per sample, was more sensitive than the former. The final PCR products were subjected to DNA‐chip‐based electrophoresis and the results were evaluated as follows: when one or two well‐defined peaks were visualized as equivalent to the positive control, the case was judged to be a monoclonal proliferation; when a smear or ladder pattern was observed, the case was considered as a polyclonal proliferation; and when no bands were observed, the case was judged to be non‐amplifiable . When monoclonal bands were observed in at least one of the tri‐parallel PCRs for the IGH rearrangement assay (tubes A–C), the case was judged to be a monoclonal proliferation.…”

“…Re‐amplification of the first‐round PCR products and nested PCR strategies are techniques that are frequently used to increase the sensitivity and specificity of PCR protocols . We recently described a novel semi‐nested modification of the standard BIOMED‐2 IGH assay and showed that tumor clonality was detected in nearly 90% of B‐cell lymphoma cases . In the present study, we devised a semi‐nested modification to the PCR protocols for the BIOMED‐2 IGK rearrangement clonality assay.…”