Immunoglobulin heavy chain gene analysis in lymphomas: a multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays

Abstract

Determination of monoclonality through an evaluation of immunoglobulin heavy chain (IgH) gene rearrangements is a commonly performed and useful diagnostic assay. Many laboratories that perform this assay do so by the polymerase chain reaction (PCR). To evaluate current methods for performing IgH gene testing, 19 different Association of Molecular Pathology (AMP) member laboratories analyzed 29 blinded B cell and T cell lymphoid neoplasm samples of extracted DNA and formalin-fixed, paraffin-embedded (FFPE) tissue and were asked to complete a technical questionnaire. From this study, it is clear that Southern blot analysis remains the diagnostic gold standard, with a 100% diagnostic sensitivity and specificity. There was, however, remarkable heterogeneity in the performance of, and results obtained from, IgH PCR assays with diagnostic sensitivity ranging from over 90% to as low as 20%, when evaluating the same specimens. Many laboratories overestimate the diagnostic sensitivity of their IgH PCR assay, and there was a significant, and under appreciated, drop-off (from 61.3% to 41.8%) in detection in paired FFPE as compared with fresh/frozen tissues. Fixation has a dramatic impact on the inability to perform the test on FFPE (43.1%) versus DNA already extracted from fresh or frozen tissue (2.8%). A number of variables that affected the outcome of IgH PCR were identified. Strategies that improved the detection of monoclonal IgH rearrangements include: the addition of FRII to the FRIII upstream primer (increasing detection from 57.3% to 73.6%) and the use of the FR3A rather than the FR3 FRIII primer (increasing detection from 54.7% to 69.7%). Although numerous variables (from DNA extraction to PCR product detection) were evaluated, making it difficult to mandate alterations in laboratory practice, these findings ought to prompt diagnostic molecular pathology laboratories to reevaluate their claims of sensitivity, as well as their methodologies. Both pathologists and surgeons need to ensure that not all submitted material is fixed, if there is adequate sample. Importantly, there is a need for greater standardization to reduce the unacceptably high false negative rate of this crucial diagnostic assay.

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Figure 1.

Immunoglobulin heavy chain gene primers. Sequences (5′→3′) of IgH primers used by the individual laboratories, ranked according to ability to detect a monoclonal IgH rearrangement in the distributed B cell lymphoma specimens. Sens; sensitivity, expressed as a percentage detection for each individual laboratory. A: FRIII primers. As compared with FR3A, FR3 has an extra six 5′ bases and one less 3′ base. The letters in the body of the table reflect nucleotide wobble in the primer sequence (Y, C/T; S, G/C; N, G/A). = identical or similar to FR3A sequence (average detection rate, 69.7%). = identical or similar to FR3 sequence (average detection rate, 54.7%). This difference in detection rate is significant (P = 0.03). B: JH primers. The letters in the body of the table reflect the variation in the primer nucleotide sequence, as compared with that designated at the top. = identical or similar to JHa sequence (average detection rate, 70.4%). = identical or similar to LJH sequence (average detection rate, 69.4%). = identical or similar to CFW1 sequence (average detection rate, 70.8%). = variant sequence (average detection rate, 30%). This difference in detection rate for the variant sequence is significant (P < 0.0001).