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


OBJECTIVE
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 kappa/lambda 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.
Catherwood
Mark A
MA
Haemato-Oncology Laboratory, Department of Haematology, Belfast City Hospital, Belfast, Northern Ireland. mark.catherwood@bll.n-i.nhs.uk
Gonzalez
David
D
Patton
Caroline
C
Dobbin
Edwina
E
Venkatraman
Lakshmi
L
Alexander
H Denis
HD
D004273:DNA, Neoplasm / Q000235:genetics; D054278:Embryonal Carcinoma Stem Cells; D015322:Gene Rearrangement, B-Lymphocyte; D018858:Germinal Center / Q000473:pathology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000276:immunology / Q000473:pathology; D008224:Lymphoma, Follicular / Q000235:genetics / Q000276:immunology / Q000473:pathology; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics / Q000276:immunology / Q000473:pathology; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000473:pathology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D027041:Somatic Hypermutation, Immunoglobulin
D004273:DNA, Neoplasm; D007143:Immunoglobulin Heavy Chains