Polymerase chain reaction-based clonality analysis of cutaneous B-cell lymphoproliferative processes

Abstract

Introduction: The differential diagnosis of B-cell lymphoproliferative processes remains a challenge for pathologists, dermatologists and oncologists, despite advances in histology, immunohistochemistry and molecular biology.

Objective: Evaluate aid and limitations of clonality analysis in the diagnosis of primary cutaneous B-cell lymphomas and B-cell pseudolymphomas.

Methods: This study included 29 cases of B-cell lymphoproliferative processes classified as primary cutaneous B-cell lymphomas (13), B-cell pseudolymphomas (6) and inconclusive cases (10) using histology and immunohistochemistry. The clonality analysis was performed by polymerase chain reaction analysis of immunoglobulin light chain and heavy chain rearrangements.

Results: DNA quality was shown to be generally poor; eight samples were inadequate for polymerase chain reaction analysis. The results showed monoclonality in eight of the primary cutaneous B-cell lymphomas and polyclonality in four of the B-cell pseudolymphomas. In addition, monoclonality was shown in two of the inconclusive cases by histology and immunohistochemistry, demonstrating the utility of polymerase chain reaction as an ancillary diagnostic tool for primary cutaneous B-cell lymphomas.

Discussion: The low quality DNA extracted from these cases demanded the use of an IgH protocol that yielded small fragments and IgK. Both methods used together improved detection.

Conclusion: Use of the two protocols, immunoglobulin heavy chain FR3-trad and immunoglobulin light chain-Kappa Biomed protocols for clonality analysis improved diagnostic accuracy.

Keywords: B-cell; Clonality; Cutaneous lymphoma; Gene rearrangement; Lymphoproliferative processes; Polymerase chain reaction.

Figure 1A

Clonality analysis of a polyacrylamide gel following electrophoresis of PCR products using the IgL-K protocol. (12) Polyclonality (broad band, similar to a smear), (13) Monoclonality (well-defined, dominant band) and (14) Not defined (absence of bands or bands without definition)

Figure 1B

DNA control: amplification of housekeeping gene fragments of 100, 200, 300 and 400 bp; lane 1: case 1 presenting a band of 100 bp; lanes 2, 3 and 4: cases 2, 3 and 4, respectively, each presenting bands of 100 and 200 bp; lane 5: case 5 showing 3 bands of 100, 200 and 300 bp

Figure 2

Clonality analysis of a polyacrylamide gel following electrophoresis of PCR products using the IgH Nizet protocol. Lane 1: positive control; lanes 2, 3, 4 and 5: polyclonal results (cases 2 and 4 in duplicate); lanes 6 and 7: monoclonal results (case 13 in duplicate); and lanes 8 and 9: undefined results (case 17 in duplicate)