BIOMED-2 Multiplex Immunoglobulin/T-Cell Receptor Polymerase Chain Reaction Protocols Can Reliably Replace Southern Blot Analysis in Routine Clonality Diagnostics
2005; Elsevier BV; Volume: 7; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60580-6
ISSN1943-7811
AutoresYorick Sandberg, Ellen J. van Gastel-Mol, Brenda Verhaaf, King H. Lam, Jacques J. M. van Dongen, Anton W. Langerak,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoTo 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. 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. 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.1Van Dongen JJ Wolvers-Tettero IL Analysis of immunoglobulin and T cell receptor genes. Part I: basic and technical aspects.Clin Chim Acta. 1991; 198: 1-91Crossref PubMed Scopus (214) Google Scholar 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 paraffin-embedded 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.2Derksen PW Langerak AW Kerkhof E Wolvers-Tettero IL Boor PP Mulder AH Vrints LW Coebergh JW van Krieken JH Schuuring E Kluin PM van Dongen JJ Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia.Mod Pathol. 1999; 12: 794-805PubMed Google Scholar 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 established. In virtually all PCR studies, only the T-cell receptor-γ (TCRG) locus was analyzed at the DNA level because of the relative structural simplicity of the gene.3Arber DA Braziel RM Bagg A Bijwaard KE Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.J Mol Diagn. 2001; 3: 133-140Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 4Shadrach B Warshawsky I A comparison of multiplex and monoplex T-cell receptor gamma PCR.Diagn Mol Pathol. 2004; 13: 127-134Crossref PubMed Scopus (20) Google Scholar PCR analysis of the T-cell receptor-β (TCRB) locus as a diagnostic test has been performed mostly on cDNA using Vβ and Cβ primers. Recently, the BIOMED-2-based TCRB gene rearrangement analysis was evaluated in a large series of well-defined samples from immature and mature T-cell malignancies and was demonstrated to be a very reliable assay.5Droese J Langerak AW Groenen PJ Bruggemann M Neumann P Wolvers-Tettero IL Van Altena MC Kneba M Van Dongen JJ Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.Leukemia. 2004; 18: 1531-1538Crossref PubMed Scopus (45) Google Scholar However, these novel BIOMED-2 multiplex PCR methods for detecting B- and T-cell clonality have yet to be validated in routine diagnostic laboratory settings. The vast majority of lymphoid malignancies encountered in the West belong to the B-cell lineage (90 to 95%). Even though B-cell clonality can be assessed by flow cytometric immunophenotyping, Ig gene rearrangement analysis is the only reliable assay for paraffin-embedded and frozen tissue biopsies. Consequently, in many routine diagnostic laboratories, TCR gene rearrangement analysis is applied to a smaller number of cases per year than Ig analysis, resulting in a lower level of experience. Because our institute is a reference center for clonality analysis in suspected T-cell proliferations, we routinely obtain many T-cell proliferation samples each year. In our study, we performed a comparative prospective study of SB-PCR Ig/TCR gene rearrangements on a series of 300 specimens consecutively obtained for routine diagnostics. In general, SB analysis was performed with optimized DNA probes for the IGH locus and the TCRB locus.6Beishuizen A Verhoeven MA Mol EJ Breit TM Wolvers-Tettero IL van Dongen JJ Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results.Leukemia. 1993; 7: 2045-2053PubMed Google Scholar, 7Langerak AW Wolvers-Tettero IL van Dongen JJ Detection of T cell receptor beta (TCRB) gene rearrangement patterns in T cell malignancies by Southern blot analysis.Leukemia. 1999; 13: 965-974Crossref PubMed Scopus (56) Google Scholar To determine B- or T-cell clonality by PCR analysis, we analyzed the IGH, TCRB, and TCRG genes. To this end, we used the well-defined and fully standardized set of oligonucleotide primers and PCR protocols of the BIOMED-2 Concerted Action BMH4-CT98-3936.8van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2485) Google Scholar, 9Sandberg Y Heule F Lam K Lugtenburg PJ Wolvers-Tettero IL van Dongen JJ Langerak AW Molecular immunoglobulin/T-cell receptor clonality analysis in cutaneous lymphoproliferations: experience with the BIOMED-2 standardized polymerase chain reaction protocol.Haematologica. 2003; 88: 659-670PubMed Google Scholar Resulting PCR products were analyzed by both heteroduplex (HD) and GeneScan (GS) analysis to evaluate the diagnostic value of these methods. Our results show that the BIOMED-2 PCR-based TCR gene rearrangement analysis is more sensitive in detecting T-cell clonality than SB analysis. In instances of discordance, PCR results demonstrated agreement with histopathological diagnosis more often than SB analysis. The higher sensitivity of PCR analysis over SB analysis also holds for IGH gene rearrangements. Based on these results, we discuss the most sensitive and efficient strategy of molecular clonality analysis. From June 2001 until February 2004, 300 DNA samples from fresh or frozen tissue samples (peripheral blood [PB], n = 110; lymph node [LN], n = 68; bone marrow [BM], n = 28; skin, n = 59; bowel, n = 7; liver, n = 3; spleen, n = 1; thyroid, n = 1; vitreous fluid, n = 15; cerebrospinal fluid (CSF), n = 3; pleural fluid, n = 2; adenoid, n = 1; brain, n = 1; and maxillary sinus tissue, n = 1) were prospectively collected from a total of 218 patients with suspected malignant lymphoproliferation. Most patients were seen and followed by physicians at the Erasmus MC, University Medical Center (Rotterdam), especially at the Departments of Hematology and Dermatology. Diagnoses were based on a combination of clinical, histological, immunophenotypical, and cytomorphological data. Patients diagnosed with a malignancy were classified according to the World Health Organization classification of lymphoid neoplasms.10Jaffe ESHN Stein H Vardiman JW World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. IARC Press, Lyon2001Google Scholar High-molecular-weight DNA from fresh or frozen tissue samples was obtained by one of two methods. In the first, DNA was extracted using a phenol-chloroform extraction-based protocol, followed by ethanol precipitation and re-solubilization in Tris-EDTA buffer.1Van Dongen JJ Wolvers-Tettero IL Analysis of immunoglobulin and T cell receptor genes. Part I: basic and technical aspects.Clin Chim Acta. 1991; 198: 1-91Crossref PubMed Scopus (214) Google Scholar Alternatively, DNA was isolated using the GenElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol. DNA (20 μg) was digested with appropriate restriction enzymes, size fractionated in 0.7% agarose gels, and transferred to nylon filters as described elsewhere.1Van Dongen JJ Wolvers-Tettero IL Analysis of immunoglobulin and T cell receptor genes. Part I: basic and technical aspects.Clin Chim Acta. 1991; 198: 1-91Crossref PubMed Scopus (214) Google Scholar The Ig and TCR gene rearrangements were detected by use of 32P random oligonucleotide-labeled probes. The IGHJ6 probe (DakoCytomation, Inc., Carpinteria, CA) was used for analyzing IGH genes in combination with BglII or BamHI/HindIII digests,6Beishuizen A Verhoeven MA Mol EJ Breit TM Wolvers-Tettero IL van Dongen JJ Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results.Leukemia. 1993; 7: 2045-2053PubMed Google Scholar whereas the TCRBJ1 and TCRBJ2 probes (DakoCytomation) were used for analyzing TCRB genes in combination with EcoRI, BglII, and BamHI/HindIII digests.7Langerak AW Wolvers-Tettero IL van Dongen JJ Detection of T cell receptor beta (TCRB) gene rearrangement patterns in T cell malignancies by Southern blot analysis.Leukemia. 1999; 13: 965-974Crossref PubMed Scopus (56) Google Scholar In a selected number of cases (n = 11), the TCRG and TCR-δ (TCRD) gene rearrangements were studied as well. For analysis of TCRG gene rearrangements, the TCRGJ13 probe was used in combination with EcoRI and Pstl digests,11Moreau EJ Langerak AW van Gastel-Mol EJ Wolvers-Tettero IL Zhan M Zhou Q Koop BF van Dongen JJ Easy detection of all T cell receptor gamma (TCRG) gene rearrangements by Southern blot analysis: recommendations for optimal results.Leukemia. 1999; 13: 1620-1626Crossref PubMed Scopus (30) Google Scholar whereas the TCRDJ1 probe was used in combination with EcoRI digests for analysis of TCRD gene rearrangements.12Breit TM Wolvers-Tettero IL Beishuizen A Verhoeven MA van Wering ER van Dongen JJ Southern blot patterns, frequencies, and junctional diversity of T-cell receptor-delta gene rearrangements in acute lymphoblastic leukemia.Blood. 1993; 82: 3063-3074PubMed Google Scholar In the case of clinical suspicion of natural killer cell lymphoma and Epstein-Barr virus (EBV) infection, the presence of (clonal) EBV genome was assessed using the XhoI probe in BamHI/HindIII-digested DNA.13Raab-Traub N Flynn K The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation.Cell. 1986; 47: 883-889Abstract Full Text PDF PubMed Scopus (744) Google Scholar All amplification reactions were performed in an automated thermocycler (model ABI 9600/9700; Applied Biosystem, Foster City, CA) according to the BIOMED-2 multiplex PCR protocol.8van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2485) Google Scholar Each 50-μl PCR reaction included 100 ng of DNA, 10 pmol of 5′ and 3′ oligonucleotide primers, 0.2 mmol/L dNTP, 5 μl of 10× buffer II (TCRB and TCRG), or 5 μl of 10× Gold buffer (IGH and IGK), and 1 to 2 U of Ampli-Taq Gold polymerase (Applied Biosystems). The concentration of MgCl2 ranged from 1.5 mmol/L (TCRB tube C, TCRG, IGH, and IGK) to 3 mmol/L (TCRB tubes A and B). The cycling parameters were as follows: pre-activation for 7 minutes at 95°C, followed by 35 cycles of 30 seconds denaturation at 95°C, >30 seconds annealing at 60°C, and >30 seconds extension at 72°C. After the last cycle, a final extension step of at least 10 minutes at 72°C was performed. For amplification of IGH rearrangements, we used six framework FR1-VH primers, seven FR2-VH primers, seven FR3-VH primers, and one carboxyfluorescein-labeled JH consensus primer in three multiplex combinations (IGH multiplex tubes A, B, and C). DNA from the precursor B-cell line NALM-6 was used as a positive control. In the case of inconclusive IGH gene results, the samples were further analyzed using multiplex PCR reactions for the Ig-κ (IGK) genes. For amplification of the IGK locus, we used seven Vκ primers, two FAM-labeled Jκ primers, one intron RSS primer, and one FAM-labeled Kde primer in two combinations (IGK multiplex tubes A and B). For amplification of TCRB rearrangements, Vβ family and FAM-labeled Jβ primers were used in two different combinations (TCRB multiplex tubes A and B) and in one combination containing Dβ and Jβ primers (TCRB multiplex tube C). DNA obtained from the following immature T-cell lines was used as a positive control: RPMI-8402 (tube A), CML-T1 (tube B), and Jurkat (tube C). For amplification of TCRG genes, we used Vγ and FAM-labeled Jγ primers in two multiplex combinations (tubes A and B).8van Dongen JJ Langerak AW Bruggemann M Evans PA Hummel M Lavender FL Delabesse E Davi F Schuuring E Garcia-Sanz R van Krieken JH Droese J Gonzalez D Bastard C White HE Spaargaren M Gonzalez M Parreira A Smith JL Morgan GJ Kneba M Macintyre EA Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2485) Google Scholar Positive controls consisted of DNA from immature T-cell lines MOLT 3, RPMI-8402 (tube A), and Jurkat (tube B). In a few cases, TCRD rearrangements were analyzed using a single multiplex tube containing Vδ, Jδ, and Dδ primers. All BIOMED-2 multiplex PCR tubes were obtained from InVivoScribe Technologies (Carlsbad, CA; www.invivoscribe.com). All cell lines are available at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Features of these cell lines have been summarized in detail elsewhere.14Drexler H The Leukemia-Lymphoma Cell Line Facts Book. Academic Press, San Diego, CA2001Google Scholar After Ig/TCR amplification, 10 μl of PCR products was loaded on 1% agarose gels to check whether PCR products had been formed. Subsequently, PCR products were further analyzed by heteroduplex and GeneScan analysis (see below) to assess whether the obtained PCR products were derived from monoclonal or polyclonal cell populations. The PCR products for HD analysis were denatured at 94°C for 5 minutes and subsequently renatured at 4°C for 60 minutes to induce duplex formation.15Langerak AW Wolvers-Tettero LM van Dongen JJM Immunoglobulin and T-cell receptor gene analysis in the diagnosis of lymphoid malignancies.Rev Clin Exp Hematol. 1997; 3: 3-27Google Scholar Afterwards, the duplexes were immediately loaded on 6% nondenaturing polyacrylamide gels in 0.5× Tris-boric acid-EDTA buffer, run at ambient temperature, and visualized by ethidium bromide staining. A 100-bp DNA ladder (Promega Corporation, Madison, WI) was used as size marker. GeneScan analysis was performed using an automated ABI PRISM 377 fluorescent sequencer (Applied Biosystems) for the majority of PCR products, whereas the remaining samples were analyzed using an ABI 3100 Genetic Analyzer (Applied Biosystems). When using the first detection method (ABI 377), 2 μl of 10× diluted PCR products were mixed with 2.0 μl of formamide, 0.5 μl of 6-carboxytetramethylrhodamine-labeled internal standard (Genescan 500-TAMRA; Applied Biosystems), and 0.5 μl of loading buffer (blue dextran). After denaturation at 95°C for 2 minutes and cooling, 3 μl of the mixture was size-separated on a high-resolution polyacrylamide gel and analyzed. The size and profile of the PCR products was determined using GeneScan Analysis software v. 2.1 (Applied Biosystems).16Linke B Bolz I Fayyazi A von Hofen M Pott C Bertram J Hiddemann W Kneba M Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.Leukemia. 1997; 11: 1055-1062Crossref PubMed Scopus (60) Google Scholar, 17Kneba M Bolz I Linke B Hiddemann W Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis.Blood. 1995; 86: 3930-3937PubMed Google Scholar For the second method (ABI 3100), 1 μl of a 5× dilution of PCR products was added to 10 μl of a MilliQ:rhodamine-labeled internal standard (GeneScan-500 ROX; Applied Biosystems) mixture (40:1). After denaturation at 95°C for 2 minutes and cooling, the samples were size-separated and detected. The size and profile of the PCR products were determined using GeneScan Analysis software v. 3.7.1. (Applied Biosystems). GeneScan analysis results in a Gaussian distribution of multiple peaks, representing many different PCR products in case of reactive lymphoproliferations but gives a single peak in case of monoclonal lymphoproliferation. Oligoclonality is defined as multiple peaks in a polyclonal background. We consecutively investigated 300 samples (from 218 patients) that were submitted for routine diagnostics. In all patients a clinical diagnosis of malignant lymphoproliferative disease was initially considered. All samples were studied by multiplex PCR-based HD and GS analysis of Ig/TCR gene rearrangements, whereas SB analysis could be performed on only 176 out of the 300 samples (Table 1). Some samples were analyzed for both Ig and TCR clonality. Overall, 258 samples were analyzed by PCR for T-cell clonality; of these samples, 150 were analyzed by TCRB SB. PCR IGH gene rearrangement analysis was performed in 87 samples, and SB analysis was performed in 48 of these samples.Table 1Summary of Performed SB- and PCR-Based Ig/TCR AnalysisSamples (patients)Clonality analysisMultiplex PCRSBTCR only214 (155)28 (24)Ig only39 (30)3 (3)Ig and TCR47 (33)145 (119)Total300 (218)176 (146) Open table in a new tab Because of our position as a reference center and our extensive expertise on TCR gene rearrangement analysis, the focus of our study lies on T-cell clonality analysis. SB and PCR analyses were, in principle, performed once. In practice this means that Ig clonality was evaluated with the IGHJ6 probe and in three different FR IGH multiplex PCR reactions. In limited cases, additional SB and PCR analysis of the IGK gene was performed. TCR clonality was analyzed with two TCRB SB probes as well as with TCRB and TCRG multiplex PCR protocols. Few samples were studied by SB analysis for TCRG/TCRD gene rearrangements or for EBV genome and PCR analysis of TCRD gene rearrangements. In case of doubtful or discrepant results, assays were repeated. Of 300 samples, 176 were analyzed simultaneously by multiplex PCR and SB analysis (Table 1). Molecular data were concordant for TCR analysis in 127 of 150 samples (85%), whereas Ig gene rearrangement analysis was concordant in 41 of 48 samples (85%). Table 2 shows the proportion of concordant SB-PCR results in polyclonal and clonal cases. The finding of a monoclonal T- or B-cell population both by SB and PCR clonality analysis strongly suggests a clonal (probably malignant) lymphoproliferation. Polyclonal gene rearrangements in both assays are indicative of a reactive (benign) lymphoproliferation. Nevertheless, it should be emphasized that the results of molecular clonality studies should always be interpreted in the context of clinical, histological, and immunophenotypical data. In 30 samples derived from 27 patients, discordances were found between multiplex PCR and SB analysis. In all of these samples, monoclonality was demonstrated by PCR analysis, whereas SB did not show evidence for clonal Ig/TCR gene rearrangements.Table 2Concordance between SB- and PCR-Based Ig/TCR Analysis in Polyclonal and Clonal CasesGene rearrangement targetSB-PCR concordanceIgTCRTotal concordance41/48 (85%)127/150 (85%)Polyclonal cases25/41 (61%)83/127 (65%)Clonal cases16/41 (39%)44/127 (35%) Open table in a new tab In 23 samples derived from 21 patients, multiplex PCR analysis demonstrated clonal TCRB/TCRG gene rearrangements, which were not detected by SB analysis. In 17 samples, the molecular findings correlated with the histopathological diagnosis of malignant T-cell proliferations (74%). (For an example of histologically proven ALCL, see Figure 1.) In the remaining samples there was no clear indication for a T-cell malignancy at the time of diagnosis (Table 3).Table 3Discordances between SB and PCR Results in Suspect T-Cell ProliferationsSBPCR procedureCase no.Sample no.Final clinicohistological diagnosis*AILT, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large-cell lymphoma; CTCL, cutaneous T-cell lymphoma; HES, hypereosinophilic syndrome; LyP, lymphomatoid papulosis; MF, mycosis fungoides; MPD-U, myeloproliferative disease, unclassifiable; PTLD, post-transplant lymphoproliferative disorder; SS, Sézary syndrome; T-ALL, T-cell acute lymphoblastic leukemia; T-LBL, T-cell lymphoblastic lymphoma; T-LGL, T-cell large granular lymphocyte leukemia; T-NHL NOS, T-cell non-Hodgkin's lymphoma, not otherwise specified.TCRBTCRGTCRDTCRBTCRGTCRD12001-146 (skin)LyPGNDNDCCND22001-148 (LN)AILTGNDNDCCND32001-152 (PB)T-LGLGGNDCCND42001-162 (skin)ALCLGGNDCCND52002-003 (LN)Peripheral T-NHL, NOSGNDNDCPP62002-022 (PB)MFGNDNDCCND72002-033 (LN)CD30+ CTCLGNDNDCCND82002-047 (PB)T-LGLGNDNDCCND92002-050 (LN)ALCLGNDGCPC102002-113 (BM)T-ALLGNDGCPC112002-144 (BM)MPD-UGGGCCP122003-022 (LN)AILTGGGCCND132003-172 (BM)T-LBLGGGCCC142003-236 (BM)HESGNDNDCPND2003-296 (PB)HESGNDNDCCND152003-245 (skin)MFGGGCPND162003-270 (PB)SSGNDNDCPND2004-007 (LN)SSGNDNDCPND172004-001 (skin)SSGNDNDCPND18†Case 18 was diagnosed with a CD30+ CTCL 10 years before.2004-004 (PB)ReactiveGNDNDCCND192004-005 (skin)CD30+ CTCLGNDNDCPND202004-011 (BM)ReactiveGNDNDCPND212004-020 (LN)ReactiveGNDNDCCNDR, rearranged; G, germline; C, clonal; P, polyclonal; ND, not done.* AILT, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large-cell lymphoma; CTCL, cutaneous T-cell lymphoma; HES, hypereosinophilic syndrome; LyP, lymphomatoid papulosis; MF, mycosis fungoides; MPD-U, myeloproliferative disease, unclassifiable; PTLD, post-transplant lymphoproliferative disorder; SS, Sézary syndrome; T-ALL, T-cell acute lymphoblastic leukemia; T-LBL, T-cell lymphoblastic lymphoma; T-LGL, T-cell large granular lymphocyte leukemia; T-NHL NOS, T-cell non-Hodgkin's lymphoma, not otherwise specified.† Case 18 was diagnosed with a CD30+ CTCL 10 years before. Open table in a new tab R, rearranged; G, germline; C, clonal; P, polyclonal; ND, not done. PCR analysis of the TCR locus identified clonal gene rearrangements in six samples derived from five patients in whom no diagnosis of T-cell leukemia or lymphoma could be made. Case 11 was diagnosed as myeloproliferative disease, unclassifiable. Case 14, diagnosed as idiopathic hypereosinophilic syndrome (HES), showed identical clonal TCR rearrangements in the BM and PB samples by PCR analysis. Immunophenotyping of the PB sample demonstrated a small T-cell population (0.3% of leukocytes) with an aberrant phenotype (CD3−/CD4+/CD5+). It has been shown that in some HES patients, abnormal monoclonal T cells can be found in PB.18Kitano K Ichikawa N Shimodaira S Ito T Ishida F Kiyosawa K Eosinophilia associated with clonal T-cell proliferation.Leuk Lymphoma. 1997; 27: 335-342PubMed Google Scholar, 19Simon HU Plotz SG Dummer R Blaser K Abnormal clones of T cells producing interleukin-5 in idiopathic eosinophilia.N Engl J Med. 1999; 341: 1112-1120Crossref PubMed Scopus (410) Google Scholar, 20Brugnoni D Airo P Rossi G Bettinardi A Simon HU Garza L Tosoni C Cattaneo R Blaser K Tucci A A case of hypereosinophilic syndrome is associated with the expansion of a CD3−CD4+ T-cell population able to secrete large amounts of interleukin-5.Blood. 1996; 87: 1416-1422PubMed Google Scholar, 21Cogan E Schandene L Crusiaux A Cochaux P Velu T Goldman M Brief report: clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome.N Engl J Med. 1994; 330: 535-538Crossref PubMed Scopus (282) Google Scholar However, in the literature so far, no clonal T-cell populations identical to the one in PB have been detected in BM. Further research should reveal the meaning of the clonal T-cell population in BM. Because patients diagnosed with idiopathic HES and associated clonal T-cell populations are at risk of developing T-cell lymphoma,19Simon HU Plotz SG Dummer R Blaser K Abnormal clones of T cells producing interleukin-5 in idiopathic eosinophilia.N Engl J Med. 1999; 341: 1112-1120Crossref PubMed Scopus (410) Google Scholar, 22Roufosse F Schandene L Sibille C Willard-Gallo K Kennes B Efira A Goldman M Cogan E Clonal Th2 lymphocytes in patients with the idiopathic hypereosinophilic syndrome.Br J Haematol. 2000; 109: 540-548Crossref PubMed Scopus (154) Google Scholar, 23O'Shea JJ Jaffe ES Lane HC MacDermott RP Fauci AS Peripheral T cell lymphoma presenting as hypereosinophilia with vasculitis: clinical, pathologic, and immunologic features.Am J Med. 1987; 82: 539-545Abstract Full Text PDF PubMed Scopus (79) Google Scholar careful follow-up is required in this case. Case 18 was admitted to our hospital in 1994 and was diagnosed with CD30+ cutaneous T-cell lymphoma. Although the patient was clinically healthy without any signs of relapse, the PB sample demonstrated monoclonal TCRB rearrangements in 2004. The PCR analysis on the paraffin-embedded skin tissue sample from 1994 demonstrated weak clonal TCRB rearrangements, identical to the ones found in the PB sample 10 years after presentation. We strongly recommend follow-up of this patient as well. A skin relapse of an ALCL was diagnosed in case 20. Staging resulted in the detection of weak clonal TCRB gene rearrangements in the BM sample, not identical to the ones found in the skin. Flow cytometry and histopathology did not demonstrate localization of malignant lymphoma. Finally, case 21 was diagnosed as reactive lymphadenopathy after extensive immunohistochemical and flow cytometric analysis of the LN sample. In seven samples derived from six patients, multiplex PCR analysis demonstrated clonal B-cell populations that were not detected by SB analysis (Table 4). IGH mo
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