BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics

Abstract

To establish the most sensitive and efficient strategy of clonality diagnostics via immunoglobulin and Tcell receptor gene rearrangement studies in suspected lymphoproliferative disorders, we evaluated 300 samples (from 218 patients) submitted consecutively for routine diagnostics. All samples were studied using the BIOMED-2 multiplex polymerase chain reaction (PCR) protocol. In 176 samples, Southern blot (SB) data were also available, and the two types of molecular results were compared. Results of PCR and SB analysis of both T-cell receptor and immunoglobulin loci were concordant in 85% of samples. For discordant results, PCR results were more consistent with the final diagnosis in 73% of samples. No false-negative results were obtained by PCR analysis. In contrast, SB analysis failed to detect clonality in a relatively high number of samples, mainly in cases of low tumor burden. We conclude that the novel BIOMED-2 multiplex PCR strategy is of great value in diagnosing patients with suspected B-and T-cell proliferations. Because of its higher speed, efficiency, and sensitivity, it can reliably replace SB analysis in clonality diagnostics in a routine laboratory setting. In most patients with suspected lymphoproliferative disorders, discrimination between reactive and malignant cell populations can be assessed by histomorphology or cytomorphology supplemented with immunohistochemistry or flow cytometric immunophenotyping. However, in 5 to 10% of patients, diagnosis is more complicated and less straightforward. In such cases, molecular gene rearrangement studies have proved useful as an additional diagnostic tool. Molecular clonality analysis is based on the fact that, in principle, all cells of a malignancy have a common clonal origin and show clonally (identically) rearranged immunoglobulin (Ig) or T-cell receptor (TCR) genes. The diagnosis of malignant B-and T-cell proliferations is therefore supported by the finding of Ig/TCR gene clonality, whereas reactive lymphoproliferations show polyclonally rearranged Ig/TCR genes. 1 Gene rearrangement analysis can be performed by Southern blot (SB)-and polymerase chain reaction (PCR)-based techniques. Despite the high reliability of SB analysis, it is increasingly replaced by PCR techniques because of the greater efficiency and sensitivity of PCR. Moreover, PCR is relatively easy, less labor intensive, and requires much less high-molecular-weight DNA. Also, SB analysis cannot be performed on paraffinembedded tissue because the isolated DNA is often degraded. Therefore, there is a strong need to replace SB analysis with reliable PCR techniques. However, PCR studies have often suffered from false-negative results due to improper annealing of primers and/or the presence of somatic hypermutation. 2 Both SB and PCR analyses of the immunoglobulin heavy chain (IGH) locus have been demonstrated to be very useful and reliable techniques in clonality assessment of suspected B-cell malignancies. However, the most useful gene target for identifying T-cell clonality is less well e...

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements

“…6 In addition, the complementary nature of BIOMED-2 TCR-␤ and TCR-␥ PCR assays in detecting clonal TCR GRs has been proven in several multicenter studies using freshly collected or frozen samples of mature T-cell neoplasms. 3,23,24 Our results, using predominantly FFPE tissue, confirm these findings. Furthermore, we believe that independently confirming either positive or negative multi-V TCR-␥ assay results with another set of mono-V TCR-␥ primers can add to the clinical robustness of the TCR-␥ PCR test.…”

“…Later validations of this PCR strategy also mainly relied on fresh/frozen tissues. 3,22,23 Since it is known that simultaneous amplification of multiple targets can reduce the sensitivity of the PCR assay for one or more targets, 17 we compared the BIOMED-2 multiplex primers that target two TCR-␥ V gene segments per reaction (multi-V) with an approach that targets a single V segment per reaction (mono-V) in a real-life clinical setting that necessitates the use of predominantly FFPE tissue. It is important to note that the mono-V primers are similar, but not identical, to the multi-V primers; which, in theory, could lead to differences in the sensitivities between the mono-V and multi-V approaches.…”

“…Degradation of DNA in paraffin-embedded tissue is another limiting factor. 23 In a recent study, PCR was actually superior to Southern blot analysis in detecting T-cell clonality. 23 On one hand, TCR-␤ analysis might be of help, but it is well known that not every case with a clonal TCR-␥ GR is accompanied by TCR-␤ clonality and vice versa.…”

“…23 In a recent study, PCR was actually superior to Southern blot analysis in detecting T-cell clonality. 23 On one hand, TCR-␤ analysis might be of help, but it is well known that not every case with a clonal TCR-␥ GR is accompanied by TCR-␤ clonality and vice versa. 3,23 Morphological information also has its flaws since one uses TCR clonality assays to solve diagnostic dilemmas raised by morphological examination.…”

Application of flow cytometric in situ hybridization assay to Epstein–Barr virus‐associated T/natural killer cell lymphoproliferative diseases

“…Multiplex PCR using the T cell Gene Rearrangement/Clonality assay (InVivoScribe Technologies, La Ciotat, France) was used to evaluate TCR gene; this assay was developed and standardized in a European BIOMED-2 collaborative study. (30,31) FISH assay. The FISH assay was performed as described previously.…”