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00019606-201006000-00002ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2010 Lippincott Williams & Wilkins, Inc.19June 2010 p 70-77Evaluation of T-cell Clonality in Archival Skin Biopsy Samples of Cutaneous T-cell Lymphomas Using the Biomed-2 PCR ProtocolOriginal ArticlesLukowsky, Ansgar PhD*; Muche, J. Marcus MD†; Möbs, Markus PhD*; Assaf, Chalid MD*; Humme, Daniel*; Hummel, Michael PhD‡; Sterry, Wolfram MD*; Steinhoff, Matthias MD**Department of Dermatology and Allergy‡Institute of Pathology, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Germany†Westfries Gasthuis Hoorn, NetherlandsReprints: Ansgar Lukowsky, PhD, Department of Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Charitéplatz 1 D-10117 Berlin, Germany (e-mail: [email protected]).The authors state no conflict of interest.AbstractRecently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) γ and TCRβ PCR as well as an in-house TCRγ PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRγ PCR revealed 81% clonality, the in-house TCRγ method revealed 86% clonality, and the Biomed-2 TCRβ revealed 78% clonality in CTCL samples generating at least the 300 bp fragment in the Biomed-2 control PCR. We found clonal TCRβ rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRγ PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRγ assay verifies our previously published findings on clonally expanded T-cells in CTCL.The DNA sequence of a T-cell receptor (TCR) gene rearrangement provides a unique marker for each individual T-lymphocyte. As in lymphomas all malignant cells are derived from a single transformed lymphoid cell, the presence of an expanded clonal TCR gene rearrangement indicates a neoplastic T-cell proliferation. Thus, its molecular analysis by polymerase chain reaction (PCR) is widely used in the diagnosis of various T-cell lymphomas (TCL), including cutaneous T-cell lymphoma (CTCL). In particular, TCR gene analysis is very supportive in those cases in which a differential diagnosis between reactive lesion and malignant lymphoma on the basis of immunohistological criteria alone is challenging. This holds particularly true for the diagnosis of CTCL consisting predominantly of small tumor cells embedded in a dense inflammatory background.In recent years, a large number of PCR assays have been designed for the detection of clonal TCR gene rearrangements. These are easier to handle and more sensitive than the previously used Southern blot methods. To date, PCR analyses of the TCRγ genes are predominantly applied in routine practice. This is based on the relatively simple TCRγ locus configuration and the large homology within the Vγ and Jγ gene segments, limiting the number of required primers. However, the limited junctional diversity also results in a high background amplification of rearrangements of reactive T-cells. Moreover, in a significant proportion of malignant proliferations, the tumor clone escapes detection. To overcome these limitations, several DNA-based TCRβ PCR protocols were developed1–3. One advantage of the TCRβ PCR is the extensive combinatorial repertoire of TCRβ rearrangements and its large hypervariable region, which results in a higher specificity. However, due to a highly degenerated consensus primer or a large number of different tubes, the efficacy and comparability of these TCRβ PCR protocols varies considerably. To improve and standardize PCR technologies, several known centers of lymphoma diagnosis and research from 7 European countries have elaborated and tested new protocols and primer sets for PCR-based clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action BMH4-CT98-3936).4 The Biomed-2 methods include 2 TCRγ PCRs and 3 PCRs for complete and incomplete TCRβ rearrangements, respectively. Compared with preexisting PCR protocols or the Southern Blot methods, it was shown that the Biomed-2 TCR assay is more sensitive in detecting clonal TCR rearrangements and that the Southern Blot is no longer regarded as “gold standard” for TCR genotyping.3,5,6 Recently, the value of the Biomed-2 protocol was confirmed by its application to a large series of the most frequent systemic mature T-cell malignancies using fresh or frozen material.7 Thus far, a representative number of formalin-fixed and paraffin-embedded (archival) samples have not been investigated. However, the use of archival material is essential, as paraffin-embedded tissue samples are supplied for routine diagnosis procedures in most instances. Furthermore, only limited data are currently available for clonality detection by the Biomed-2 TCR assay in lesional skin biopsies of CTCL.8,9To evaluate the reliability and applicability of Biomed-2 methods in archival tissue of CTCL, we applied the Biomed-2 TCRγ and TCRβ assay to 107 archival paraffin-embedded tissue samples of 84 CTCL patients, 3 systemic TCL patients (sTCL) and 20 controls. The results of the Biomed-2 TCRγ approach4 were compared with our in-house TCRγ assay,10,11 which has been used in CTCL diagnosis in our department for more than 10 years, routinely.MATERIALS AND METHODSPatients and Clinical SamplesA total of 107 consecutively collected formalin-fixed and paraffin-embedded lesional skin biopsies obtained from 84 CTCL patients, 3 cases of sTCL, and 20 controls (1 sample per case) were investigated. All diagnoses were based on clinical, histologic and immunohistologic criteria. According to the WHO-EORTC classification,12 the CTCL patients were diagnosed with the following: 56 mycosis fungoides (MF), that is, 18 patch, 35 plaque, and 3 tumor stage; 14 lymphomatoid papulosis (LyP); 6 cutaneous anaplastic large TCL (cALCL); 5 Sézary syndrome (SS); and 3 pleomorphic CTCL (pleoCTCL). The sTCL were 2 cases with cutaneous lesions of an angioimmunoblastic TCL and one patient with a peripheral TCL, unspecified. Samples from 20 patients without a malignant T-cell proliferation (5 cutaneous B-cell lymphoma, 13 benign inflammatory dermatoses and 2 pseudolymphoma) served as controls. The research committee of the Charité-Universitätsmedizin Berlin approved the described studies. Informed consent for the experimental studies was obtained from the patients. The study was conducted according to the Declaration of Helsinki Principles.T-cell LinesSix clonal human T-cells lines, that is, MyLa, SeAx, Jurkat, Molt-4, Peer, and HH, were investigated by the Biomed-2 PCR and also used as clonal controls. Jurkat and Peer cells were additionally applied for evaluation of analytical sensitivities.DNA Preparation and Control PCRGenomic DNA was prepared manually from all samples by the same standard procedure using paraffin extraction with Roticlear and proteinase K digestion as described earlier.11 The quality of each DNA sample was confirmed by Biomed-2 control PCR4 yielding amplicons of 100, 200, 300, and 400 bp in size in a multiplex assay and by subsequent electrophoresis on a 1.5% agarose gel followed by staining with GelRed Nucleic Acid Gel Stain (Biotium, Hayward). As recommended for the subsequent Biomed-2 TCR PCRs, only DNA samples that provided at least a faint 300 bp band were used.13 For estimation of the detection limits in formal-fixed specimens, serial dilutions of DNA from Jurkat and Peer cells in tonsillar DNA were analyzed. For this purpose, cell pellets derived from 108 cells of the respective cell lines were generated by centrifugation and, after removal of the supernatants, the cell pellets were fixed in neutral buffered formalin (4%) for 2 hours and, subsequently, embedded in paraffin. Normal tonsillar tissue was fixed for approximately 24 hours under standard conditions using 4% neutral buffered formalin. DNA was obtained from these materials and dilution series were prepared.TCR PCRThe DNA of the skin biopsy samples was amplified by the 2 Biomed-2 TCRγ PCR (sets A and B) and 3 TCRβ PCR (sets A, B, C) according to the original Biomed-2 report.4 The in-house TCRγ PCR method was performed for the same set of samples as described earlier.11 The TCRγ PCRs 1 and 2 were applied to all samples, whereas the TCRγ PCR 3 amplifying rearrangements of Vγ with Jγ1.1/2.1 (JP1/2) gene segments were only applied to lymphoma samples that were nonclonal in the PCRs 1 and 2. All primers used in this study were purchased from a commercial local supplier (BioTez, Berlin, Germany). The primer synthesis includes a standard purification by gelfiltration but not by HPLC. Each reaction was screened by electrophoresis on a 1.5% agarose gel followed by staining with GelRed before fluorescence fragment analysis (FFA, see below) was applied. Each TCR PCR showing a clonal outcome in the FFA (see below) was repeated to confirm the individual clonal TCR fragment length and to exclude pseudoclonality. Moreover, all TCR PCRs of CTCL samples showing a polyclonal result were verified by at least one replicate. The McNemar test was performed for the statistical evaluation of selected PCR results. Significance was assumed at a confidence level of at least 90%. Fluorescence fragment analysis.After PCR and a positive screening on the agarose gel, products were subjected to FFA on the ABI 310 PRISM capillary sequencing instrument using the Gene Mapper 3.7 software (Applied Biosystems, Weiterstadt, Germany). A successful PCR from DNA of polyclonal T-cells displayed approximate Gaussian profiles fitting the relevant size ranges. Peak height ratios were applied for the assessment of the FFA from all TCR PCRs14: clonality was assumed if 1 peak dominated the fluorescence intensity profile providing a peak-height ratio of at least 2. The peak height ratio was calculated by dividing the clonal peak height by the mean height of the left and right adjoining peaks as recorded by the Gene Mapper program. Moreover, the height of the suspected clonal peaks had to exceed the mean height of all polyclonal background peaks generated in the given set. As a T-cell clone can exhibit up to 2 rearrangements of the TCRγ locus,3 patterns with more than 2 dominant amplification products are not compatible with a single clonal T-cell population.RESULTSThe lymphoma samples, controls and T-cell lines were tested for T-cell clonality with the 3 PCR methods (in-house TCRγ, Biomed-2 TCRγ, Biomed-2 TCRβ). An assay was evaluated as positive if at least one of the various PCR tubes of the method in question revealed a clonal PCR product. Exemplary clonal and nonclonal (polyclonal) profiles of the Biomed-2 PCR are shown in Figures 1 and 2. When compared with profiles of products of fresh/frozen material, the same types of curves were received with peaks within the expected size ranges.4 The results are presented in detail in Table 1 and are summarized in Table 2.JOURNAL/dimp/04.03/00019606-201006000-00002/table1-2/v/2021-02-17T200020Z/r/image-tiff Analysis of the CTCL and sTCL Samples: ResultsJOURNAL/dimp/04.03/00019606-201006000-00002/table2-2/v/2021-02-17T200020Z/r/image-tiff Analysis of the CTCL, sTCL, and Control Samples: SummaryJOURNAL/dimp/04.03/00019606-201006000-00002/figure1-2/v/2021-02-17T200020Z/r/image-jpeg Fragment profiles of Biomed-2 TCRγ PCR products (examples). A, tube A; B, tube B; left, monoclonal; right, polyclonal.JOURNAL/dimp/04.03/00019606-201006000-00002/figure2-2/v/2021-02-17T200020Z/r/image-jpeg Fragment profiles of Biomed-2 TCRβ PCR products (examples). A, tube A; B, tube B; C, tube C; left, monoclonal; right, polyclonal.Using serial dilutions with DNA from formalin-fixed cell lines and formalin-fixed tonsils, detections limits of approximately 2.5% to 5% were demonstrable when different primer sets were applied for the detection of TCRγ and TCRβ rearrangements.Results of the PCR AssaysThe in-house TCRγ assay showed 72/84 clonal CTCL samples (frequency of clonality/diagnostic sensitivity: 86%). The highest rates of T-cell clonality (100%) were observed in SS, cALCL, and pleoCTCL, and the lowest rate (79%) in LyP. In MF, 47/56 (84%) clonal specimens were detected, with a higher rate of clonality in the plaque stage (89%) than in the patch stage (78%). One of the 3 tumor stage samples remained nonclonal. All of the 3 sTCL and all 5 T-cell lines were found to be clonal, and all of the controls were nonclonal.The Biomed-2 TCRγ assay detected 68/84 clonal CTCL samples (81%). Again, all specimens derived from SS, cALCL, and pleoCTCL were found to be clonal, whereas the lowest rate of T-cell clonality was observed in LyP (71%). In MF, 44/56 (79%) clonal specimens were detected, with a higher rate of T-cell clonality in the plaque stage (83%) than in the patch stage (72%). One of the 3 tumor stage samples remained nonclonal. All 3 sTCL samples were found to be clonal. One of the 20 control samples exhibited T-cell clonality with a small (uncertain) peak of 147 bp in Biomed-2 TCRγ tube A indicating a V10-Jγ1.3/2.3 (Jγ1/2) recombination. This sample was obtained from a patient with a cutaneous marginal zone lymphoma. All T-cell lines investigated were found to be clonal, and the corresponding TCRγ and TCRβ amplicon sizes were in line with previous descriptions, with one exception.15 These are shown in Table 3. In Molt 4 cells we obtained a slightly different result: set A presented a biallelic TCRγ rearrangement of 219/242 bp, whereas a value of 223/242 bp was quoted.15 The Biomed-2 TCRβ found 62/80 clonal CTCL specimens (78%), that is, 56/80 (70%) in the VJ-PCR (sets A and B) and 40/80 (50%) in the DJ-PCR (set C). Four CTCL samples (1 cALCL, 3 LyP) could not be analyzed, because all material was used for the TCRγ assays. The highest rates of T-cell clonality were found in SS (100%), pleoCTCL (100%), and cALCL (80%), the lowest rate (73%) in LyP. In MF, 42/56 (75%) samples revealed clonal PCR products, with lower rates in patch than plaque stages (72% vs. 77%). The TCRβ assay also failed to detect T-cell clonality in the one MF tumor stage sample that was nonclonal in both TCRγ assays. T-cell clonality was detected in 2/3 sTCL. Two of 20 control samples obtained from patients with pseudolymphoma showed small clonal peaks (clonality uncertain). Both of these were nonclonal in all other TCR PCRs.JOURNAL/dimp/04.03/00019606-201006000-00002/table3-2/v/2021-02-17T200020Z/r/image-tiff Biomed-2 TCR PCR in Clonal Human T-cell Lines: ResultsDespite the fact that the TCRβ assay is highly complex, the TCRβ VJ PCR (sets A and B) failed completely to generate specific products in only 4/80 CTCL samples. In the β DJ assay (set C) from 8/80 samples, no products were formed. Amplification in all three sets failed in only three cases, thereby indicating the high applicability of this assay to DNA from paraffin-embedded tissues. Interestingly, the TCRβ DJ PCR did not indicate T-cell clonality in any of the investigated CTCL cases exclusively, as in each of the samples with a clonal TCRβ DJ rearrangement at least one further Biomed-2 PCR revealed a clonal product.Comparison of the TCRγ AssaysCompared with the Biomed-2 TCRγ assay, the in-house TCRγ assay showed a slightly higher diagnostic sensitivity (86% vs. 81%); however, the difference is not yet significant (confidence level: 89%). As shown by the control samples, both methods revealed a high specificity.In a subset of 11 CTCL samples, that is, 8 MF (4 patch, 3 plaque, 1 tumor stage) and 3 LyP, both the Biomed-2 TCRγ PCR and the in-house TCRγ PCR unanimously did not detect clonal rearrangements. Both methods revealed clonality in all 3 sTCL specimens.Comparison of the Biomed-2 TCRγ and TCRβ AssaysWe showed that the diagnostic sensitivities of both PCR methods were comparable to each other (81% vs. 78%). Interestingly, 5 CTCLs (2 MF patch, 1 MF plaque, 2 LyP) were nonclonal in the Biomed-2 TCRγ assay but exhibited clonal TCRβ rearrangements. In contrast, 7 CTCLs (2 MF patch, 3 MF plaque, 1 LyP, 1 cALCL) were nonclonal or nonamplified in the Biomed-2 TCRβ assay but could be shown to be clonal in the Biomed-2 TCRγ assay. Owing to the partially different outcome, the combination of both assays enhanced the diagnostic sensitivity significantly towards 87% (confidence level: 96.3%).Combining all PCR AssaysBy combining all TCR PCR assays applied in our study, clonality was shown in 76 of the CTCL cases (90%), whereas 8 specimens (6 MF-3 patch, 2 plaque, 1 tumor stage, and 2 LyP) remained nonclonal.DISCUSSIONTo date, PCR analysis of rearranged TCR genes in combination with high-resolution electrophoresis has been used commonly, and represents an important technique in the diagnosis of CTL including CTCL. Since 1989, a large number of different PCR protocols for the detection of clonally rearranged TCR genes have been applied in lymphoma diagnosis and research. In addition, many different electrophoretic techniques such as heteroduplex polyacrylamid gel electrophoresis and FFA have been used for subsequent PCR product analysis. Therefore, it was difficult to compare results from different reports or laboratories. In view of this fact, a comprehensive panel of standardized PCR methods has been developed in cooperation within the European Biomed-2 project.4 Owing to the high reliability of the Biomed-2 primer protocol in fresh or frozen material of systemic lymphomas and to the lack of data on its applicability in paraffin-embedded tissue and in CTCL, we applied the Biomed-2 TCR assay to a representative number of archival skin samples of patients with various CTCLs. The results were compared with our in-house PCR assay, which we have applied for more than 10 years. A similar evaluation of the Biomed-2 IgH-PCR in archival samples of patients with cutaneous B-cell lymphoma has already been performed.16In our study investigating paraffin-embedded CTCL tissues, Biomed-2 TCRγ PCR and in-house TCRγ PCR revealed comparable diagnostic sensitivities of 81% and 86%, respectively. With the Biomed-2 TCRβ assay, 78% of CTCL cases were found to be clonal. These results show similarity to a small series of 10 archival MF samples (including 5 cases of MF tumor stage) showing clonality in 80% of the cases using the Biomed-2 protocol.9 Either comparable or slightly lower detection rates have also been stated in numerous earlier reports using various other TCRγ assays in paraffin-embedded tissues9,17. Assaf et al1 revealed T-cell clonality in 100% of 24 archival CTCL samples applying a seminested consensus TCRβ PCR/FFA. However, in contrast to our study, only advanced stages of disease were investigated. Moreover, nested and seminested assays are particularly prone to generating pseudoclonal results, and require repeated analyses of each clonal DNA sample.To our knowledge, the only published study applying the Biomed-2 protocols in CTCL detected clonal rearrangements in 73% by the Biomed-2 TCRγ and in 62% by the Biomed-2 TCRβ assay with subsequent FFA. Only fresh/frozen samples were used.8 Recently, Ponti et al17 showed T-cell clonality in 84% of MF cases (with approximately 70% to 76% in the early stages) and in 100% of SS cases applying a different TCRγ PCR protocol and FFA to 203 frozen skin samples. A slightly lower percentage of clonal MF cases was detected when using heteroduplex polyacrylamide gel electrophoresis for PCR product separation.17 Fairly similar to our results, Morgan et al18 found 85% of clonally expanded TCRγ rearrangements and 75% of clonally expanded TCRβ rearrangements when applying the Biomed-2 protocols to a group of 20 CTCLs (17 MF, 3 SS). In that study, DNA was prepared from both fresh and paraffin-embedded skin sections, but the portions of both materials were not quoted.18 Our data from paraffin-embedded CTCL tissues are also similar to those from fresh or frozen samples. It should be emphasized that all samples generated the 300 bp fragment in the Biomed-2 control tube. On this condition, the lower integrity of DNA as expected after extraction from paraffin obviously did not reduce the diagnostic sensitivity of the PCR-based clonality analysis. Thus, even the more complex Biomed-2 TCRβ assay revealed comparable frequencies of T-cell clonality. However, a higher complexity of the PCR assay may result in amplification of pseudoclonal PCR products, resulting in a lower diagnostic specificity. If this holds true for the Biomed-2 assays can only be evaluated with more control samples. The complexity of the PCR primers may also explain the differences between the in-house TCRγ assay and the Biomed-2 TCRγ assay: by splitting the investigation of the possible TCRγ rearrangements into 3 PCRs, the in-house assay revealed slightly more clonal rearrangements in CTCL (Table 2); however, the difference is not significant. In fact, the Biomed-2 TCRγ assay failed to detect T-cell clonality in 5 CTCLs in which clonality was shown by the in-house PCR. Conversely, the Biomed-2 TCRγ PCR exclusively showed clonality in one case. The in-house approach and the Biomed-2 test revealed almost identical analytical sensitivities of 2.5% to 10% in formalin-fixed material, depending on the T-cell line. We have received equivalent detection thresholds with dilutions of DNA from freshly collected clonal T-cells applying the TCRγ Biomed-2 protocol19 or in-house PCR.14 Thus, the different results of the TCRγ assays are caused by different primer binding positions, rather than by different analytical sensitivities.Insufficient priming of the TCR gene segments due to germ line configuration, or to incomplete, deleterious or trans-rearrangements may explain the nonclonality of 8 CTCL cases in all PCR assays. In general, clonal rearrangements are not detectable using TCRγ PCR in at least 10% of lesional skin samples from CTCL, regardless of the PCR and electrophoresis method applied.20 Priming at different genes explains the differences between the TCRγ and TCRβ analyses. Here, the consistency is lower when compared with the differences in the TCRγ assays. Combining the Biomed-2 TCRγ and TCRβ methods provided significantly increased diagnostic sensitivity. Therefore, the Biomed-2 TCRβ assay (with the exception of the TCRβ DJ PCR, which did not exclusively indicate a clonal rearrangement) should supplement the TCRγ tests in routine CTCL analysis. This is in line with a recent analysis of 188 fresh frozen samples of systemic T-cell malignancies, in which only 4.3% of the clonal rearrangements were detected by the TCRβ assay.7 This procedure in clonality testing has already been recommended by others3 and, as shown here, applies also to paraffin-embedded samples. In this investigation, the combination of all TCR tests used enhances the frequency of detected T-cell clonality in CTCL to 90%. In the PCR-based clonality analysis of DNA from archival samples, a substantial percentage of clonal cases may be missed. Christensen et al21 found, in 4/18 sTCL cases (22%), clonal TCRγ rearrangements only with frozen and not with paraffin-embedded tissue; however, they did not use the Biomed-2 methods or a TCRβ assay.In comparison with an earlier report,15 we repeatedly received a slightly smaller amplicon for one rearra-nged allele of Molt 4 cells in the Biomed-2 TCRγ test (219 vs. 223 bp). The difference seems to be too large for a normal variation of FFA sizing, and may be caused by genetic aberrations occurring during long-term maintenance of the cell line.Using the Biomed-2 assays, we received an “uncertain-clonal” outcome of 3 control samples. Accordingly, a combination of these assays could reduce the diagnostic specificity; however, more controls have to be investigated for a reliable statement. This matter is of special concern as a clonal result of the assay is used as meaningful supplementary information to confirm a malignant lymphoproliferation. Nevertheless, due to the fact that the presence of a clonal TCR rearrangement does not always indicate malignancy, it has to be emphasized that the accurate integration of clinical, histomorphologic, immunohistochemical, and molecular biologic data is still mandatory. In conclusion, our data underline the reliability and applicability of the standardized Biomed-2 primers and protocols, and show their applicability to the diagnosis of paraffin-embedded skin biopsies of CTCL. The Biomed-2 TCRγ PCR and in-house TCRγ PCR revealed similar diagnostic sensitivities and specificities. However, the Biomed-2 TCRγ protocol is currently highly recommended for routine analysis of CTCL, in particular in achieving a standardization of TCR PCR techniques. This permits much better data comparability and exchange of experience in TCL/CTCL diagnosis. Moreover, the in-house TCRγ PCR is more laborious, requiring a third tube set. As shown here, the Biomed-2 TCRβ method may be helpful, particularly in cases suspected of having CTCL without detectable clonal TCRγ rearrangement. The Biomed-2 methods are also qualified to reliably detect oligoclonal expansions and clonal heterogeneities in CTCL. However, the confirmed diagnostic sensitivity and specificity of our in-house TCRγ assay, routinely used in CTCL diagnosis in our laboratory for more than 10 years, verifies our previously published findings on clonally expanded T-cells in CTCL.10,22REFERENCES1. Assaf C, Hummel M, Dippel E, et al. High detection rate of T-cell receptor beta chain rearrangements in T-cell lymphoproliferations by family specific polymerase chain reaction in combination with the GeneScan technique and DNA sequencing. Blood. 2000;96:640–646.[Context Link][CrossRef][Medline Link]2. Hodges E, Krishna MT, Pickard C, et al. Diagnostic role of tests for T cell receptor (TCR) genes. 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