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 T-cell 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. Just as with SB results, PCR results should always be interpreted in the context of clinical, immunophenotypical, and histopathological data.

Figure 1

Skin localization of ALCL in case 4. A: Histology of ulcerating skin tumor. Skin biopsy specimen demonstrates localization of dermal infiltrate of large cells with irregular nuclei (HE; magnification, ×100). Note the large hallmark cells (inset) (HE; magnification, ×600). B: The large neoplastic cells are strongly positive for CD30 (magnification, ×200). Note the strongly stained membrane and the dot-like staining in the Golgi complex area (inset) (magnification, ×600). C: The neoplastic cells also stain for CD4 (magnification, ×200). D: PCR-based GeneScan analysis. Bi-allelic monoclonal TCRG gene rearrangements could be identified in skin DNA.

Figure 2

Polymorphic B-cell PTLD-associated disease in case 27. A: Histology of LN. There was no recognizable lymph node architecture (HE; magnification, ×200). There were numerous blasts (inset) (HE; magnification, ×600) intermingled with an infiltrate of smaller lymphocytes. B: EBER-ISH showed scattered positively stained nuclei, indicating the presence of EBV RNA (magnification, ×400). C: CD3 staining of the predominantly small T cells. Occasionally, larger cells stained positively (magnification, ×400). D: CD20 stained the majority of the large blasts. Note the numerous mitotic figures (arrows) (magnification, ×400). E and F: PCR-based GeneScan analysis of LN DNA. Oligoclonal TCRB gene rearrangements could be identified (E), whereas a monoclonal IGH gene rearrangement was detected (F).

Figure 3

Strategy for BIOMED-2 multiplex PCR TCR clonality analysis in suspect T-cell proliferations. In TCRαβ⁺ or TCR-negative T-cell proliferations, there is no clear evidence-based preference for either the TCRB or TCRG locus as the initial target for clonality diagnostics. Thus, either TCRB or TCRG can be used as the first line target, followed by the other locus as the second target. For TCRγδ⁺ T-cell proliferations, the TCRG locus is the clear first line target for clonality assessment, followed by TCRD as the second target. In the absence of clonal TCR gene rearrangements in PCR, SB analysis might still be considered for all suspect T-cell proliferations, provided that enough high-quality DNA is available. Although the PCR strategy as described here is suitable for fresh or frozen tissue samples, it could possibly be applied to DNA isolated from paraffin-embedded tissue samples, provided that DNA quality is such that PCR products of ∼300 bp can be amplified.