Optimal primer selection for clonality assessment by polymerase chain reaction analysis: I. Low grade B-cell lymphoproliferative disorders of nonfollicular center cell type

Abstract

Recent polymerase chain reaction (PCR)-based studies focused on the detection of immunoglobulin heavy chain gene (IgH) rearrangements have suggested that clonal populations may be amplified more easily from certain categories of B-cell neoplasia than others and that primer makeup can be a critical factor in successful amplification. However, these particular reports contained relatively few low grade B-cell lymphoproliferative disorders of nonfollicular center cell type (LG-BLPD) and used only a limited panel of available primer sets for PCR amplification of monoclonal B-cell populations. To address this issue more extensively we evaluated 156 samples of LG-BLPD by the PCR to determine optimal primer selection in this setting. All cases were classified according to standard morphological and immunophenotypic criteria, with monoclonality documented by Ig light chain restriction analysis. The LG-BLPD included 33 cases of chronic lymphocytic leukemia (CLL), 57 cases of small lymphocytic lymphoma (SLL), 10 cases of atypical CLL, 32 cases of mantle cell lymphoma (MCL), 17 plasma cell neoplasms (PCNs), and seven cases of hairy cell leukemia (HCL). All primer sets included a 3′ IgH joining region consensus primer, whereas the 5′ IgH variable region (VH) primer was different in each set. The first-line panel included the following: Set 1, VH-framework III consensus primer, and Set 2, seven separate VH-framework I family-specific primers. A reserve panel of alternate VH consensus primers directed at framework II or III regions was used only when Set 1 showed no evidence of B-cell monoclonality. Overall, monoclonal B-cell populations were amplified in 153 of 156 (98%) neoplasms by the PCR; two PCNs and one plasmacytoid SLL were negative with all primer pairs. Higher amplification efficiency was obtained with Set 1 primers than Set 2 primers in the entire series (96% ν 84%) as well as within each subtype of LG-BLPD. In each diagnostic category monoclonal B-cell populations were detected by Set 1 in 96% or more of neoplasms, the only exception being plasma cell neoplasms where the monoclonality detection rate was 76% (ie, 13 of 17 cases). With the additional use of Set 2 and the reserve panel primer sets, 15 of 17 (88%) PCNs were shown to contain PCR-detectable monoclonal B-cell populations. Our data suggest that the vast majority of CLLs, SLLs, MCL, and HCL samples can be successfully genotyped by the PCR with a single primer set (Set 1). In contrast, plasma cell tumors and rare SLL samples appear to require more extensive analysis (ie, additional primer sets) to adequately assess B-cell clonality.