Detection of monoclonality in low‐grade B‐cell lymphomas using the polymerase chain reaction is dependent on primer selection and lymphoma type

Abstract

Detection of B-cell monoclonality using the polymerase chain reaction (PCR) promises the quick and cost-effective separation of monoclonal from polyclonal B-cell disease. However, the efficiency of the method has yet to be fully assessed, particularly with regard to disease type and selection of PCR primers. We have evaluated two approaches based on amplification of the immunoglobulin heavy chain gene using framework 2 (Fr2) and framework 3 (Fr3) region primers. Frozen tissue samples from 94 cases of low-grade B-cell lymphoma were investigated, all of which had previously been shown to be monoclonal by Southern blot analysis. Using a Fr2 primer, we were able to show monoclonality in 85 per cent of cases; with Fr3, 80 per cent of cases; and using both techniques in separate reactions, 90 per cent of cases. Thus, a significant false-negative rate exists with either primer which can be reduced by using both. We also found a difference in the efficiency of detection in different types of lymphoma; only 87 per cent of mucosa-associated lymphomas and centroblastic/centrocytic lymphomas were shown to be monoclonal, whereas all of the other lymphoma types tested were positive using one or both methods. We conclude that PCR detection of B-cell monoclonality allows rapid analysis of tissue samples, including paraffin-processed material. False-negative results which occur in some types of lymphoma can be reduced by the use of two or more primer combinations.

Immunoglobulin Mutational Status Detected through Single-Round Amplification of Partial VH Region Represents a Good Prognostic Marker for Clinical Outcome in Chronic Lymphocytic Leukemia

“…The clonality detection rate of V H FR2 consensus primer was previously reported between 58% and 85% in B-cell malignancies. [41][42][43][44][45][46] Most studies, using PCR assay to detect clonality, examined heterogeneous collections of B-cell neoplasms, and thus did not specify the actual monoclonality detection rates in the different subtypes of B-cell lymphoma or leukemia. The sensitivity of PCR assay can vary among different categories of B-cell malignancies due to a different rate of somatic mutations and to preferential IgV H usage; in particular B-CLL seems more amenable to PCR clonality detection than other B-cell neoplasms.…”

Diffuse blastoid B-cell lymphoma: a histologically aggressive variant of t(14;18)-negative follicular lymphoma

“…2 Amplification of the immunoglobulin heavy chain gene from both framework 2 and framework 3 part of the V segment to the J region was carried out using seminested PCR protocols, as described by Diss et al 3 The T-cell receptor gamma (TCR-g) chain gene was amplified using the method described by McCarthy et al 4 After PCR amplification, 10 ml of each PCR product was run on 10% polyacrylamide mini-gels for 1 h at 125 V, which were then stained with ethidium bromide and viewed under UV light.…”

Intravascular bone marrow accumulation in persistent polyclonal lymphocytosis: a misleading feature for B-cell neoplasm

“…Amplified products (from 240 to 280 bp) were loaded on an 8% polyacrylamide gel. 16 For the study of T-cell receptor g genes rearrangement, a one-step seminested PCR was performed using three primers. Two were directed against variable V regions, respectively, Vg11: 5 0 TCT GG (G/A) GTC TAT TAC TGT GC 3 0 and Vg101: 5 0 CTC ACA CTC (C/T) CA CTT 3 0 , and one against the junction region.…”