Correlation of PCR-Detected Clonal Gene Rearrangements with Bone Marrow Morphology in Patients with B-Lineage Lymphomas

Abstract

Bone marrow biopsy is the conventional staging and posttherapy evaluation method for assessing marrow involvement by lymphoma. Polymerase chain reactions (PCR) for antigen receptor rearrangements have the potential to increase the detection of minimal degrees of marrow involvement. The present study is a concurrent morphologic and PCR evaluation of 225 staging or posttherapy marrow biopsies from 127 patients with B-lineage non-Hodgkin's lymphoma. The biopsies were morphologically categorized into four groups: group 1 (positive for lymphoma), 60 biopsies (27%); group 2 (suspicious for lymphoma), 20 biopsies (9%); group 3 (lymphocytic lesions of indeterminate biology), 22 biopsies (10%); and group 4 (negative for lymphoma), 123 biopsies (54%). Molecular studies were performed on concurrently obtained aspirates and used consensus immunoglobulin-heavy-chain (IgH) and IgH/bcl-2 gene PCR primers. A molecular clone was detected in 53 of the 225 aspirates (24%): group 1, 34 aspirates (57%); group 2, five aspirates (25%); group 3, one aspirate (5%); and group 4, 13 aspirates (11%). A PCR-positive aspirate was present in 47% of follicular lymphomas, 58% of diffuse large cell lymphomas, and 72% of the other lymphomas in the group I specimens. Morphology or PCR was positive in 79 of the 225 cases (35%). The molecular detection of clonality in the aspirate DNA from cases with positive morphologic findings was lower than anticipated. The discordance between morphology and PCR results may be related to sample variation between the trephine biopsy and aspirate, a failure to aspirate sufficient lymphoma cells, or insufficient primer homology for amplification. DNA extracted from trephine sections may provide results more concordant with morphology, because PCR detected a clone in 10 of 11 DNA specimens extracted from trephine biopsies with positive morphologic findings and PCR negative aspirates.

Benign lymphoid aggregates in the bone marrow: distribution patterns of B and T lymphocytes

“…PCR for immunoglobulin heavy-chain gene rearrangement may help distinguish benign from malignant bone marrow lymphoid aggregates by demonstrating clonality [21,22], although false-negative results have been encountered in some of these studies [22,23]. Others have advised caution in the use of PCR and DNA sequence analysis because they may have a high falsepositive rate in benign aggregates, particularly in patients with associated immune-mediated disorders [24].…”

Immunoglobulin heavy chain gene analysis in bone marrow biopsies and corresponding lymph node specimens: Dependency on pre-treatment, histological subtype and extension of B-cell lymphoma

“…Therefore the method presented here is clearly superior to the strategy of others examining the BMB solely for morphological analysis and smears for further molecular clonality analysis (18,24,25).…”

“…However, their major drawback is an increased false-negative rate. Possible technical reasons for false-negative results have been reviewed (18,19). In certain clonal populations, despite optimal reaction conditions, the consensus primers may lack sufficient homology to anneal with the pertinent IgH regions because of somatic hypermutation or ongoing mutations.…”

“…Other reasons include competitive amplification of DNA from rearranged reactive bystanders, interfering chromosomal translocations or deletions. Although increasing the number of amplification cycles may enhance detection in some specimens, excessive cycling can result in increased polyclonal back- ground smearing and nonspecific bands that complicate assay interpretation, because the polyclonal smear may mask a monoclonal band or a nonspecific band may mimic a clonal band (18). Quantitative mRNA analysis of light chain expression via real-time PCR assay is well suitable for the analyses of highly mutated germinal centre/post-germinal centre lymphoma (20).…”

Association of Clinical Status of Follicular Lymphoma Patients after Autologous Stem Cell Transplant and Quantitative Assessment of Lymphoma in Blood and Bone Marrow as Measured by SYBR Green I Polymerase Chain Reaction

“…In this patient, the 1-kb nested PCR product of BCL2/JH was sequenced. A more 3Ј primer, MBRH, 26 was paired with JH, generating a product through RQ-PCR that was smaller (300 bp) and likely more efficiently amplified. .…”