Application of BIOMED-2 clonality assays to formalin-fixed paraffin embedded follicular lymphoma specimens: Superior performance of the IGK assays compared to IGH for suboptimal specimens

Abstract

The BIOMED-2 PCR-based immunoglobulin gene rearrangement assays have quickly become the most commonly used laboratory method for detection of B-cell clonality. Therefore, the reliability of these assays under various conditions has become increasingly important. When studying paired cases of follicular lymphoma (FL) from individual patients, we used these assays to assess clonality in 40 formalin-fixed paraffin-embedded (FFPE) specimens from 19 patients diagnosed with FL. The assays of IGH rearrangement failed to give a clonal result in 26/40 (65%) specimens, while the IGK assays failed in only 3/40 (8%) specimens. The high failure rate of the IGH assays for this set of FFPE lymphomas cannot be explained by systematic problems with DNA extraction or amplification because the same IGH assays resulted in a low failure rate (3/32, 9%) for FFPE small lymphocytic lymphoma/chronic lymphocytic leukemia specimens and for fresh frozen FL specimens (1/6, 17%). Furthermore, in a second validation set of 13 FFPE follicular lymphoma the failure rate was 9/13 (69%). Therefore, the BIOMED-2 IGH assay did not perform well on FFPE follicular lymphoma specimens, and the IGK assay may be superior for assessing clonality when no fresh/frozen tissue is available.

Expanded and highly active proliferation centers identify a histological subtype of chronic lymphocytic leukemia ("accelerated" chronic lymphocytic leukemia) with aggressive clinical behavior

“…4 Clonality was assessed by studying immunoglobulin heavy chain gene rearrangement from peripheral blood and paraffin-embedded tissue samples following BIOMED-2 protocols in 12 patients. 25 …”

The value of molecular diagnostics in primary cutaneous B-cell lymphomas in the context of clinical findings, histology, and immunohistochemistry

“…22 Especially in systemic follicular lymphomas and PCFCL, Igk analyses are superior to BIOMED-2 for IgH. 22,28 The reason for this might be the lower rate of SHM in the Igk locus and that the average size between the PCR products is smaller in Igk BIOMED-2. 12,20,28 However, both monoclonal results of our benign lymphoid infiltrates were achieved with Igk analyses (tube A).…”

“…12,27 However, all samples were archival skin specimens, which are more difficult to analyze than freshly prepared samples. 12,23,28 The reason for this could be DNA degradation, which is more common in older specimens especially if they are formalin fixed, which causes irreversible DNA changes.…”

Malleable Immunoglobulin Genes and Hematopathology – The Good, the Bad, and the Ugly

“…Adding IGK@ PCR to IGH@ PCR results in a dramatic improvement in the detection of clonality in follicular lymphomas, increasing the yield by up to 60%. 42,43,48 A number of possible explanations have been proposed for this phenomenon, 48 including the following: i) IGK@ PCR includes the use of a Kde primer, to amplify a unique deletion found at this locus, and which is not subjected to SHM 10 ; ii) even a functional IGK@ VJ rearrangement may be subjected to less SHM than an IGH@ rearrangement 51 ; iii) the product size of the most frequently amplified IGK@ V-segment is relatively small (ϳ150 bp), allowing for enhanced amplification compared with the larger FR1 and FR2 IGH@ products; and iv) one of the IGH@ genes will likely have been "consumed" in the prototypic t(14;18) of follicular lymphoma, rendering only one IGH@ locus (compared with potentially two IGK@ loci) amenable to analysis.…”