Detection of clonality using PCR of the antigen receptor genes has a relatively infrequent but occasionally critical niche in the diagnostic workup of non-Hodgkin lymphomas (NHL). The application of PCR-based clonality detection for Hodgkin lymphomas is much less developed but its role could be more critical than it is for NHL because, in contrast to the workup of NHL, flow cytometry does not detect the clonality in HL. Here we show that about half of all classic HL FFPE specimens can be expected to show B cell clonality using a combination of the BIOMED-2 IGK and IGH assays.
The two cases presented here indicate scenarios in which detection of clonality using DNA purified from FFPE tissue contributed substantially to the diagnosis. In the first case the diagnosis was unsuspected due to the unusual presentation and strong clinical suspicion of a sarcoma. The infradiaphragmatic presentation, and direct bone involvement are quite unusual sites of presentation of de novo HL7
. In the second case, the diagnosis was made difficult by the small size of the specimen, the syncytial malignant cells which mimicked a NHL, and the lack of classic immunophenotype.
Only a handful of publications have described PCR-based clonality detection for HL using non-microdissected specimens.3, 5, 8-10, 4
Just 3 of these have assessed the use of the standardized BIOMED-2 primer system for FFPE HL specimens (). The sensitivity for detecting clonality in HL in all of these studies is similar, and clearly lower than that reported for most other histologic subtypes of lymphoma.4
There are several reasons that the PCR-based methods are less sensitive for HL compared to NHL. First, the malignant cells in HL are often relatively sparse. Second, there may be an abundance of non-malignant B cells which produce a polyclonal background signal that lowers the analytical sensitivity for detection of the clonal signal. Third, the RS cells appear to subject their immunoglobulin loci to a high degree of somatic hypermutation increasing the probability that a primer binding site(s) will be altered causing the PCR to fail. Nevertheless, several groups have shown the successful application of the PCR-based BIOMED-2 clonality assays to HL ().
Summary of Published Studies of Clonality in HL using BIOMED-2 Methodology Applied to FFPE Specimens.
The sensitivity of the BIOMED-2 assays depends on the histology, the specimen type, and the specific assay.6
For example, the BIOMED-2 IGH assays have very high sensitivities when applied to small lymphocytic lymphoma (SLL) specimens, whether these specimens are fresh/frozen or FFPE. In contrast, the same assays fail for about 50% of FFPE follicular lymphoma (FL) specimens while showing >90% sensitivity for fresh FL specimens. The dependence on the choice of immunoglobulin locus is also exemplified by FFPE FL specimens: while the sensitivity is about 50% with IGH assays, the IGK assays have a sensitivity of >90%.11
Although several recent publications on antigen receptor PCR in Hodgkin lymphoma employed the BIOMED-2 primer sets, our study differed in using heteroduplex analysis on polyacrylamide gels rather than Genescan analysis which involves the use of a fluorescent primer and separation by capillary electrophoresis.12
Both methods were described in the initial BIOMED-2 publication and were reported to have similar sensitivities for the IGK and IGH assays.6
In the heteroduplex procedure, the PCR products are denatured and then allowed to re-anneal. The likelihood of any specific DNA product re-associating with its exact complement in a polyclonal pool is extremely small. Therefore, the products derived from polyclonal B cells form a heteroduplex in which there are a small number of mismatches. The mismatches cause the annealed products to have a lower mobility in polyacrylamide gel electrophoresis. The specific amplicon from a clonal B-cell population is much more abundant and reassociates to form homoduplexes without mismatches. The heteroduplex signal derived from the polyclonal B cells appears at a higher apparent molecular weight easing the visualization of the signal derived from the clonal population. This is shown in , which compares the results with and without the heteroduplex procedure for IGK PCR. Without the use of the heteroduplex procedure a small clone (10% +, 5% +) could not be seen in the strong polyclonal signal. Using the heteroduplex procedure the clone is easily appreciated. Laboratories that have compared the Genescan and heteroduplex methods have noted a preference for heteroduplexing in analysis of the IGK locus due to its restricted junctional diversity, in contrast to a slight advantage in sensitivity for the Genescan method for the other antigen receptor genes.2, 6
For HL we found a similar overall clinical sensitivity compared to the other studies () and conclude that the heteroduplex analysis provides an adequate and cost effective alternative to approaches based on capillary electrophoresis.
Image 5 Heteroduplex Analysis of the IGK locus. The IGK PCR analysis was done as described in Materials and Methods, except that for the lanes labeled N the PCR products were electrophoresed after PCR without the use of the heteroduplex procedure. The lanes labeled (more ...)
Chute et al3
studied clonality in FFPE HL specimens using the BIOMED-2 IGH assays and found about 25% of HL cases to be clonal. These investigators found that the likelihood of clonality detection with the IGH assay increased with greater numbers of CD30+ cells. We also found a relationship between the density of CD30+ forms and clonality detection using the IGH assay but there was not any suggestion of a similar effect of CD30+ cells on the sensitivity of the IGK assay (). This result suggests that the IGK assay is less dependent on the number of malignant cells, perhaps in part explaining its relatively increased sensitivity in detecting clonality in HL where the malignant cells are often sparse.
Chute et al3
also found that the rate of positive results with the IGH assay was inversely proportional to the density of non-malignant B cells, a finding that we did not reproduce. While we included plasma cells as a component of the polyclonal background and Chute et al did not, we do not feel that this difference is large enough to explain the disparity. We suspect that this difference may be explained by the heteroduplex step in our procedure which reduces interference from the background signal due to polyclonal B cells. As shown in , the heteroduplex method allows clonal populations as small as 5% to be clearly detected in a polyclonal background.
Our finding that PCR can demonstrate clonality in about 50% of CHL without microdissection is unlikely to be explained by “pseudoclonality”. First, sequencing of the IGK PCR products showed that all represent bona fide rearrangements of the IGK locus rather than spurious non-specific reaction products. Second, all reactions were in duplicate and gave identical clonality results, precluding false positives due to random mispriming in an early PCR cycle. Third, flow cytometry was performed on 20/23 CHL specimens positive for B cell clonality; no suggestion of B cell clonality was detected in any of these 20 specimens indicating that a “composite” lymphoma is unlikely in any of these cases.
The BIOMED-2 IGK assays may be generally more robust for clonality detection in FFPE samples from malignancies in which the IGH loci are more heavily affected by somatic hypermutation, such as follicular lymphoma and HL.13, 14
Our data showing the relatively greater robustness of the IGK compared to the IGH assay for HL are compatible with those recently suggested in a smaller study of 8 specimens ().5
We previously showed that the IGH assays failed in about 50% of FFPE follicular lymphoma specimens in which the IGK assays succeeded. Although the IGK assays are appreciably more sensitive, the IGH FR2 and FR3 assays contribute substantially to the overall sensitivity for CHL FFPE specimens. Thus, a clinical lab will need the IGK and IGH FR2/3 assays to achieve the best sensitivity in the evaluation of the occasional specimen of Classic Hodgkin lymphoma which does not show typical clinical or histologic features.