Pitfalls in TCR gene clonality testing: teaching cases
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Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.
KeywordsTCR Clonality Pitfall GeneScanning Lymphoma
Cancer cells are the progeny of a single malignantly transformed cell, and consequently, these cells are clonally related. Hence, monoclonality is a key feature of malignant tumor cell populations, which enables discrimination from oligoclonal or polyclonal, reactive processes. Clonality assessment is thus an important tool in the diagnosis of malignant lymphoproliferations, even though clonality does not always imply malignancy because some reactive processes contain large clonal lymphocyte populations. Establishment of the clonal relationship between multiple lesions at distinct locations or over time is another useful application in cancer diagnostics. In lymphoid malignancies, clonality detection is relatively straightforward due to the ample availability of highly polymorphic DNA markers, i.e., the rearrangements in the genes encoding the antigen receptors in B and T lymphocytes, the immunoglobulin (Ig) and T-cell receptor (TCR) genes, respectively [1, 2, 3, 4]. During early lymphoid differentiation, genes encoding the Ig and TCR molecules are formed by stepwise rearrangement of variable (V), diversity (D), and joining (J) gene segments. This process is referred to as V(D)J recombination [3, 4]. During this recombination process, nucleotides are deleted and randomly inserted at the joining sites, resulting in an enormous diversity of unique antigen receptors. As Ig/TCR gene rearrangements occur sequentially in the earliest stages of lymphoid differentiation, they are present in almost all immature and mature lymphoid cells [5, 6, 7]. Since lymphomas and leukemias are derived from a single malignantly transformed lymphoid cell, the tumor cells of virtually all lymphoid malignancies contain, in principle, an identical clonal IGH and/or a clonal IGK/IGL rearrangement in B-cell lymphoproliferations and an identical clonal TCRG and/or clonal TCRB gene rearrangement in T-cell lymphoproliferations. Heterogeneity in Ig/TCR rearrangements thus identifies the presence of polyclonally activated cells, as opposed to identically rearranged Ig or TCR genes that reflect a monoclonal lymphoid cell population.
In this report, we address several technical and immunobiological pitfalls especially related to TCR clonality and its detection in lymphoid malignancies.
T-cell clonality testing
Quality parameters for clonality testing
To ascertain reproducible clonality assessment and to avoid misinterpretation of the data, DNA-quality, controlled DNA-input and tissue representativity, are essential quality parameters to be evaluated in the routine diagnostic setting .
Testing the quality of the extracted DNA from the pathological specimen is an important parameter, particularly when using formalin-fixed and paraffin-embedded (FFPE) tissues. The quality of the extracted DNA from FFPE tissues might be poor, such that the TCR gene targets are not efficiently amplified. DNA quality should therefore always be checked in a control PCR analysis in which a range of differently sized PCR products from nonpolymorphic genes are amplified . The BIOMED-2 primers are designed and validated for DNA-clonality analysis from fresh or fresh frozen tissues. Although the primers have not been universally accepted for FFPE tissues, many laboratories use the BIOMED-2 primers for FFPE samples as well. Indeed, when extracted DNA from FFPE samples shows amplification up to 300 bp of the control gene PCR, TCR clonality testing using the BIOMED-2 primers for TCRB and TCRG is very well feasible. These FFPE samples have a high detection rate of clonality comparable to that from fresh frozen samples [Section 10 in 14] (personal observation). However, when the DNA is fragmented and shows amplification of 200 or even only 100 bp of the BIOMED-2 control gene PCR, the TCRB-gene targets (due to the amplicon size of 240–285 bp in tubes A and B, and 170–210 and 285–325 bp in tube C) may not efficiently be amplified and therefore potentially yielding false-negative results [Section 10 in 14]. The TCRB GeneScan profiles from fragmented DNA samples may thus be difficult to interpret and may give ambiguous results. Therefore, we do not recommend testing TCRB gene rearrangement in the case of fragmented DNA samples. For the cases showing fragmented DNA, the TCRG-gene target is more useful and reliable as the multiplex PCRs for TCRG gene rearrangements yield lower amplicon sizes between 80 and 255 bp. The detection rate of T-cell clonality using the TCRG gene as target on DNA from fresh frozen cases still is 89% .
Controlled DNA input is also an important quality parameter because the lack of both monoclonal and polyclonal signals, or nonreproducible patterns in a given sample might be explained by too low or too high amount of target DNA in the sample, the former due to a low number of T-lymphocytes in the tissue, the latter due to PCR-inhibiting factors in the extracted DNA. The latter might particularly occur in FFPE-derived DNA samples that are obtained by DNA-extraction methods without further purification. The input of DNA per PCR is important to be evaluated in order to be sure that sufficient DNA originating from target T-lymphoid cells is analyzed and thus to confirm clonality results. In addition, it is important to exclude pseudo-clonality, which may occur in the case of low input of target T-lymphoid cells. In general, 100 ng DNA is used per PCR (50 ul) for DNA samples obtained from snap-frozen tissues. For FFPE-samples, we recommend a standard DNA-extraction procedure using an affinity-purification step to get rid of small (degraded) DNA strands. Because of the probability of PCR-inhibiting factors in the sample, two concentrations, 50 and 200 ng DNA per PCR (50 ul), are used. Adjustment of the DNA input may be necessary when there is a low percentage (5–10%) of T cells present in the tissue sample. The DNA input in these cases is maximally increased to 400 ng/PCR. To quantitate DNA, affinity purification of DNA followed by spectrophotometrical DNA-concentration measurement is recommended.
A third important parameter is tissue representativity. The number of T lymphocytes and the number of suspect malignant T cells, should be checked by histological examination of the same tissue material that was used for DNA extraction (HE staining of the slides directly prior to and following the DNA extraction slides) by an experienced hematopathologist and/or supported by flow cytometry data whenever available.
Furthermore, we advise for any clonality testing to include not only monoclonal cell lines [14, 26], but also polyclonal samples such as peripheral blood mononuclear cells showing the typical polyclonal pattern of TCR gene products with a heterogeneous junctional size distribution. Such polyclonal control samples act as control for primer quality as well. In addition, samples without rearranged Ig/TCR genes (e.g., the HeLa epithelial cell line) can be informative to identify nonspecific bands/peaks that are sometimes found in multiplex assays . These three types of control samples facilitate the interpretation of patterns in the clinical samples.
Interpretation of T-cell clonality data: teaching cases
Pitfalls in TCR clonality testinga
Bands/peaks just outside size range
Junctional regions/junctions outside 5–95% size range interval
Accept as true rearrangement product; in case of doubt, sequence for confirmation
Internal deletion in, e.g., V segment
Potential rearrangement product; confirm by sequencing
Extended amplification from downstream J
Potential rearrangement product; confirm by sequencing
Multiple clonal signals
Bi-allelic rearrangements; multiple rearrangements per allele or biclonality
Consider the number of potential rearrangements per allele and per locus and judge whether this fits with clonality or biclonality
Lack of clonal signal and lack of polyclonal Gaussian curve
Few T cells in sample
Check T cell presence by histology or flow cytometry
Low DNA input/PCR
Check DNA concentration
Poor DNA quality
Check DNA quality in control PCR
Selective amplification and pseudoclonality, due to low level of specific template
Few T cells in sample
Repeat PCR in multiplicates (same tissue, second independent DNA isolation, and/or related tissue) → compare patterns for consistency
Oligoclonal T-cell repertoire in PB of especially elderly individuals
Incomplete immune system, due to, e.g., immunosenescence
Repeat PCR in multiplicates (same tissue, second independent DNA isolation, and/or related tissue) → compare patterns for consistency and compare with primary process
Oligo-/monoclonality in histologically reactive lesion
Exaggerated immune response with dominant specificity
Repeat PCR in multiplicates (same tissue, second independent DNA isolation, and/or related tissue) → compare patterns for consistency
(Re)evaluate histopathology → note that large germinal centers in the tissue may contain dominant T-cell clone(s)
Generation and technical interpretation of GeneScan clonality profiles
In the clonality profiles of the two cases (Figs. 6 and 8), it should be noted that the Y-axes are adjusted so that all histograms appear similar, with the strongest peak near the top of the histogram. It is clear that some gene rearrangement amplifications are less abundant than others. The BIOMED-2 group is aware that in the multiplex PCR tubes primers have different PCR efficiencies. A good example is the TCRB-tube B, which is less efficient compared to the other TCRB tubes and therefore will always give weaker PCR products. Because of the different PCR efficiencies, every PCR tube should be interpreted individually (using adjusted Y-axis), making use of the normal Gaussian distribution seen in polyclonal pattern of the specific target.
A dominant clonal product in a polyclonal background of cells is seen in some of the targets, like the TCRG-tube A, which assesses the Vγ1f and Vγ10 rearrangements (Fig. 6a), and the TCRB-tube C, which assesses the incomplete DJ joinings of the TCRB locus (Fig. 6b). Precise measuring peak intensities or area under the peak is not the way to interpret clonality results. The BIOMED-2 group argues against strictly quantitative approaches that compare the size (or area under the peak) of the seemingly dominant peak with neighboring peaks in PCR reactions with a significant polyclonal background, simply because the multiplex PCRs are not strictly quantitative and because such quantitative approaches are influenced by the size of the junctional regions of the clonal peaks. Short and long junctional regions more likely lead to a “clonal” diagnosis than intermediately sized junctional regions. Therefore, the different BIOMED-2 multiplex PCRs should not be interpreted using an exact algorithm and quantitative rules. For interpretation of the clonality profiles, it is, of course, evident that the clonal peak should dominate clearly above the polyclonal background (as illustrated in Fig. 6a for TCRG-tube A). Instead of using strict quantitative rules, however, we strongly recommend the use of an “inverse” approach in which immunohistological information and preferably also flow cytometric immunophenotyping information about the size of the entire lymphocyte population and the suspect malignant lymphocyte population are used to interpret the profile and the distribution of the peaks. The dominant peak in TCRG-tube A (Fig. 6a) may fit to tumor tissue containing 80% T cells of which at least 10% were suspected to be malignant. The dominant clonal peak in tube A would not fit, for example, to a tumor tissue containing 80% T cells, all of which were suspected to be malignant. Note that both the multiplex PCRs as well as the estimation of the total number of T lymphocytes and the number of suspect malignant cells are not strictly quantitative. However, it is clear that the clonality profile and the percentage of suspect malignant cells are related, and therefore, it is important to use the available immune-histological information on the percentage of T lymphocytes and the percentage of suspect malignant T cells for the final evaluation of the molecular result. We are aware that some molecular laboratories apply a strictly quantitative approach for clonality assessment, without consulting the pathologist for the histopathological information. We argue against such practical approach because a strictly quantitative approach can easily miss clonal processes or, more dangerously, can erroneously assess “clonality”. It is better to use the(immuno)histopathological information, the knowledge of the PCR target and to apply the multiple target TCR approach (TCRB and TCRG) and evaluate the entire profile of gene rearrangements, in order to reach a correct interpretation of the clonality results.
Repeating the PCR analysis might provide extra information. In diagnostic studies in the case of ambiguous results or in the case of an isolated target, repeated analysis using the same sample, a second independent DNA extraction from the same sample and/or a related sample is necessary to evaluate consistency of results. Note that ambiguous results (results that are not reproducible or difficult to interpret) are mostly derived from DNA samples from FFPE samples. The TCRB-C tube that corresponds to the TCRB-DJ incomplete rearrangements may be such an example. For this target, a polyclonal sample should show two Gaussian curves, with a first set of peaks around 180 bp representing the Dβ2 rearrangements and a second set of peaks around 300 bp representing the Dβ1 rearrangements. The Dβ1 and Dβ2 gene rearrangements can be well evaluated in cases that show good amplification. However, in other cases, the quality of DNA samples may be insufficient to amplify the larger Dβ1 products, not allowing a proper judgment of this PCR because a potentially clonal target in these cases may remain undetected. Also, upon diagnostic requests of cutaneous NHL or skin biopsies with low numbers of T cells, we strongly recommend analyzing a second, independent tissue block from a different site whenever available or at least ask for follow-up of the patient. Repeated analysis of a related but independent sample is essential to check for consistency of the pattern.
With respect to expected size ranges of PCR products that are mentioned in protocols for a given TCR target, it is important to realize that those represent the 5–95 percentiles of the natural heterogeneity of the junctional region [14, 27]. Hence, bands or peaks just outside this size range can be interpreted as true rearrangement products, even without formal proof via sequencing. When the products are considerably smaller (undersized) or larger (oversized), they still can represent true rearrangements, but then sequencing analysis is warranted for confirmation.
Not all lymphoma cases need to be evaluated for clonality assessment because most cases can be diagnosed based on morphology and immunohistology supplemented by translocation detection or cytogenetics. Molecular clonality assessment is generally performed on 10–15% of the lymphoma cases, the percentage being dependent on the experience of the hematopathologist. In our setting of clonality diagnostics, approximately 5% of evaluated cases give problems in the interpretation of molecular TCR-clonality results. The difficulties in these cases are severe degradation of DNA and, consequently, greatly decreased PCR efficiencies resulting in ambiguous results or the interpretation of a small clonal peak in a high polyclonal background, seen in an isolated target. For the latter, our strategy is to assess a second, independent tissue block from a different site whenever available or ask for follow-up of the patient.
Heteroduplex analysis or GeneScanning
Heteroduplex analysis is another tool to analyze the PCR products for discrimination between monoclonal lymphoid cells with identical junctional regions and polyclonal lymphoid cells with highly diverse junctional regions. Heteroduplex analysis uses double-stranded PCR products and takes advantage of the length and composition of the junctional regions, whereas in GeneScanning, single-stranded PCR products are separated in a high-resolution gel or polymer according to their length only (Fig. 3). The heteroduplex technique is rapid, simple, and cheap and has a detection limit of 5–10% [10, 11]. The detection limit is influenced by the frequency of polyclonal lymphocytes because the formation of many heteroduplexes will also consume a part of the monoclonal PCR products . Since heteroduplex analysis separates PCR products on the basis of junctional diversity in addition to PCR product size, it is particularly useful in the analysis of loci with restricted junctional diversity like that for the TCRG-gene rearrangements. For the TCRB-gene rearrangements, heteroduplex analysis is particularly useful for the TCRB PCR tubes A and C (the TCRB VDJ and the incomplete DJ rearrangements, respectively) and slightly less for tube B (the other set of TCRB VDJ gene rearrangements) because this multiplex PCR is less efficient compared to the other TCRB tubes. For this reason, the TCRB PCR tube B will always give weaker PCR products, which can less easily be detected using heteroduplex analysis because this approach is less sensitive compared to GeneScanning. GeneScanning is rapid and relatively simple, but needs expensive equipment. GeneScanning is generally more sensitive than heteroduplex analysis and can reach sensitivities of 1–5% of clonal lymphoid cells. We prefer the use of GeneScanning because of its sensitivity. In addition, the precise determination of the size of the PCR product can potentially be used for nonquantitative monitoring of the clonal proliferation during follow-up of the patient.
Multiple clonal rearrangements
Knowledge of the TCR loci, immunobiology, and the pitfalls of the multiplex approach are needed for proper interpretation of multiple clonal PCR products from one of the TCR gene targets.
A complex interpretation pitfall concerns the question whether detection of multiple clonal signals is equivalent to biclonality. Although biclonal malignancies do occur, first, several immunobiological and technical explanations for multiple clonal signals should be considered. Since lymphocytes carry two chromosomes, biallelic rearrangements are more common than biclonality. Second, J segment clusters such as in the TCRB locus (but also in the IGH, IGK loci) might give rise to additional “clonal” signals caused by differently sized PCR products derived from the same rearrangement. Third, the TCRB locus has a more complex configuration in that two rearrangements can occur simultaneously on the same allele, mounting up to a total of four rearrangements per T-cell clone: a V–Jβ1 or Dβ1–Jβ1 rearrangement can be followed by a Dβ2–Jβ2 rearrangement on the very same allele . Taken together, all these phenomena illustrate that, in the case of multiple clonal signals, biclonality is more of an exception to the rule.
Clonality interpretation should also be performed in the context of immunobiology. A well-known pitfall concerns the detection of oligoclonality or even (low level) monoclonality of T lymphocytes under certain immunobiologic conditions [29, 30, 31, 32, 33, 34, 35]. Detection of an oligoclonal T-cell repertoire in elderly individuals should be considered as a potential sign of an involuting immune system due to aging of the system (immunosenescence) . Repeated analysis using the same sample, a second independent DNA extraction form the same sample and/or a related sample need to be evaluated for consistency of the results. In true reactive lesions, oligoclonality or even monoclonality may reflect an exaggerated immune response with a dominant immunospecificity. Apart from repeated molecular analysis, a careful combined (re)evaluation of the clinical, histopathologic, and molecular findings is needed to correctly interpret oligo-/monoclonality results.
Interactive interpretation model
Knowledge about technical and immunobiological pitfalls of TCR rearrangement analysis is of utmost importance, but it does not suffice in all cases. For appropriate interpretation of the molecular data, it is essential to combine these data with data from immunohistology and preferably also with the results from flow cytometric immunophenotyping. Such an interactive interpretation model, with regular contacts between clinical molecular biologists, pathologists, hematologists, and immunologists guarantees integration of all available data to reach the most reliable diagnosis. Especially, the percentage of suspect malignant lymphocytes and the percentages of normal (reactive) B and T lymphocytes, as available from histopathology and/or flow cytometry, are important parameters to be considered for estimating the relevance of the TCR clonality findings. Thus, the finding of weak clonal signals (in one or more targets) in a background of polyclonal signals is hardly compatible with a large suspect cell population, and hence, such TCR findings should be interpreted with caution. On the other hand, absence of both monoclonal and polyclonal TCRG patterns in a sample showing a large T-cell infiltrate is not compatible and should lead to further analysis of other TCR targets before definitive conclusions can be drawn. Finally, the interpretation of clonality can also be dependent on the clinical presentation. Regular discussions of cases in multidisciplinary patient meetings should avoid the aforementioned misinterpretations.
This review is based on the results and discussions with many colleagues all over Europe in the BIOMED-2 Concerted Action “PCR-based clonality detection in suspect lymphoproliferations” (BMH4-CT98-3936). We thank Margarethe van Altena, Annemiek van Raaij, Paul Rombout (Radboud University Nijmegen Medical Centre, Nijmegen), and Ingrid LM Wolvers-Tettero, Ellen J van Gastel-Mol, Monique ECM Oud, Brenda Verhaaf (Erasmus MC, Rotterdam) for technical assistance.
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