ORIGINAL ARTICLE

Improved clonality assessment in germinal centre/post-germinal centre non-Hodgkin’s lymphomas with high rates of somatic hypermutation

Mark A Catherwood¹, David Gonzalez², Caroline Patton³, Edwina Dobbin⁴, Lakshmi Venkatraman⁵ and H Denis Alexander¹

¹ Haemato-Oncology Laboratory, Belfast City Hospital, Belfast, Northern Ireland
² Department of Haemato-Oncology, BLB, Institute of Cancer Research, Surrey, UK
³ Schools of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland
⁴ Biological Sciences, University of Ulster, Coleraine, Northern Ireland
⁵ Department of Histopathology, Royal Victoria Hospital, Belfast, Northern Ireland

Correspondence to:
Correspondence to:
Dr M A Catherwood
Haemato-Oncology Laboratory, Department of Haematology, Level C, Belfast City Hospital, Belfast BT9 7AB, Northern Ireland; mark.catherwood{at}bll.n-i.nhs.uk

Background: PCR detects clonal rearrangements of the Ig gene in lymphoproliferative disorders. False negativity occurs in germinal centre/post-germinal centre lymphomas (GC/PGCLs) as they display a high rate of somatic hypermutation (SHM), which causes primer mismatching when detecting Ig rearrangements by PCR.

Aims: To investigate the degree of SHM in a group of GC/PGCLs and assess the rate of false negativity when using BIOMED-2 PCR when compared with previously published strategies.

Methods: DNA was isolated from snap-frozen tissue from 49 patients with GC/PGCL (23 diffuse large B cell lymphomas (DLBCLs), 26 follicular lymphomas (FLs)) and PCR-amplified for complete (VDJH), incomplete (DJH) and Ig/ rearrangements using the BIOMED-2 protocols, and compared with previously published methods using consensus primers. Germinal centre phenotype was defined by immunohistochemistry based on CD10, Bcl-6 and MUM-1.

Results: Clonality detection by amplifying Ig rearrangements using BIOMED-2 family-specific primers was considerably higher than that found using consensus primers (74% DLBCL and 96% FL vs 69% DLBCL and 73% FL). Addition of BIOMED-2 DJH rearrangements increased detection of clonality by 22% in DLBCL. SHM was present in VDJH rearrangements from all patients with DLBCL (median (range) 5.7% (2.5–13.5)) and FL (median (range) 5.3% (2.3–11.9)) with a clonal rearrangement.

Conclusions: Use of BIOMED-2 primers has significantly reduced the false negative rate associated with GC/PGCL when compared with consensus primers, and the inclusion of DJH rearrangements represents a potential complementary target for clonality assessment, as SHM is thought not to occur in these types of rearrangements.

Abbreviations: DLBCL, diffuse large B cell lymphoma; FL, follicular lymphoma; FR, framework; GC/PGCL, germinal centre/post-germinal centre lymphoma; SHM, somatic hypermutation

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