INTRODUCTION: Cutaneous T-cell lymphoid infiltrate can represent reactive lesion or a malignant T-cell lymphoma. However, clinical and histopathological appearance can overlap in both groups with a risk of misdiagnosis. Aberrant expression of T-cell markers is not always applicable and T-cell receptor (TCR) gene rearrangement is not always accessible and diagnosis in borderline cases can be challenging. AIMS: Several types of TCR antibodies are currently available with limited knowledge of their expression in different cutaneous lymphoid infiltrates. Aim of the study is a comparison of expression of TCR antibodies in benign and malignant lymphoid infiltrates and their utility in borderline cases. METHODS: Representative cases of reactive and malignant lymphoproliferations were collected. Separate group of lesions with borderline morphology was selected for comparison. Immunohistochemical expression of TCR-V-betaF1 (TCRBF1), TCR-C-beta1 (TCRJOVI.1), TCR gamma/delta (TCRGD) and TCR delta (TCRD) was performed in all cases. TCR gene rearrangement evaluation was performed in all cases using PCR BIOMED-2 assay. RESULTS: Benign lymphoid infiltrates were all negative in TCRD and TCRGD. Expression of TCRJOVI.1 was seen in 3/10 cases and TCRBF1 in one. T-cell lymphomas were positive for TCRBF1 and TCRGD in 60% and 30% of cases respectively. TCR gene rearrangement was confirmed in 90% of lymphoma cases. All benign lesions were polyclonal. Morphologically borderline lesions showed expression of TCRBF1 in 6/10 cases and TCR gene rearrangement in 4/10 cases. Re-evaluation of the cases and clinical correlation led to the change of the diagnosis and confirmation of T-cell lymphoma in 4/10 cases. CONCLUSIONS: Expression of TCRBF1 and TCR-gene rearrangement was significantly associated with malignant infiltrates. TCRBF1 positivity in borderline cutaneous lymphoproliferations can raise the suspicion of malignancy but confirmation by TCR gene rearrangement and careful clinical correlation is still advisable.

• Klíčová slova
• Cutaneous lymphoma, Cutaneous lymphoproliferation, Inflammatory dermatosis, TCR rearrangement,
• MeSH
• antigeny CD7 analýza   MeSH
• diferenciální diagnóza MeSH
• dospělí MeSH
• genová přestavba T-lymfocytů * MeSH
• imunohistochemie * MeSH
• kožní T-buněčný lymfom genetika   imunologie   patologie   MeSH
• kůže imunologie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfoproliferativní nemoci genetika   imunologie   patologie   MeSH
• prediktivní hodnota testů MeSH
• protilátky imunologie   MeSH
• receptory antigenů T-buněk alfa-beta analýza   genetika   MeSH
• receptory antigenů T-buněk gama-delta analýza   genetika   MeSH
• senioři MeSH
• specificita protilátek MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny CD7 MeSH
• protilátky MeSH
• receptory antigenů T-buněk alfa-beta MeSH
• receptory antigenů T-buněk gama-delta MeSH

The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

• MeSH
• body zlomu chromozomu MeSH
• Equidae krev   genetika   metabolismus   MeSH
• genová přestavba T-lymfocytů * MeSH
• geny TcR * MeSH
• homologní rekombinace MeSH
• karyotyp MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty cytologie   MeSH
• malování chromozomů MeSH
• prasata genetika   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

VDJ recombination is a key process in T-cell receptor (TCR) and immunoglobulin (Ig) molecules development. Comparison of ENSEMBL and GenBank database information revealed major differences in dog T-cell receptor beta (TRB) region annotations. ENSEMBL based genomic alignment of dog TRB sequence with human sequence and annotation showed a very similar structure of TRB. However, there is only one cluster of DJC segments in dogs. In dog, 38 V segments are followed by 1 D segment, 6 J segments and 1 C segment. Like humans and mice, dogs have another V segment opposite in orientation downstream of the C segment. V segments anticipated were analyzed using the RT-PCR and capillary electrophoresis. Thirty-one of them were identified in samples of thymus and spleen RNA and thus believed to be subjected to chromosomal rearrangement and RNA splicing. We identified and analyzed probable structure of canine TCR beta region, which is different when compared to sequences published in GenBank or ENSEMBL databases.

• MeSH
• databáze genetické MeSH
• DNA primery genetika   MeSH
• druhová specificita MeSH
• genová přestavba - beta řetězec receptoru antigenů T-buněk MeSH
• geny TcR beta * MeSH
• lidé MeSH
• myši MeSH
• psi genetika   imunologie   MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• sestřih RNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• psi genetika   imunologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA primery MeSH

Rearrangements of T- and B-cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high-throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high-throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V- and J-segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T-lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies.

• Klíčová slova
• T-cell receptor alpha chain, acute lymphoblastic leukaemia, clonal rearrangements, high-throughput sequencing, lymphoproliferative disorders,
• MeSH
• DNA nádorová genetika   MeSH
• genová přestavba - alfa řetězec receptoru antigenů T-buněk * MeSH
• hematologické nádory genetika   MeSH
• lidé MeSH
• lymfoproliferativní nemoci genetika   MeSH
• multiplexová polymerázová řetězová reakce * MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA nádorová MeSH

The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a "public" repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.

• MeSH
• antigenní specifita receptorů T-buněk genetika   MeSH
• dvojčata monozygotní genetika   MeSH
• genetická variace genetika   MeSH
• genová přestavba T-lymfocytů genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• receptory antigenů T-buněk fyziologie   MeSH
• rekombinace genetická MeSH
• sekvence nukleotidů MeSH
• stárnutí genetika   imunologie   MeSH
• vývojová regulace genové exprese genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

• MeSH
• akutní lymfatická leukemie genetika   MeSH
• genetické markery genetika   MeSH
• genová přestavba T-lymfocytů genetika   MeSH
• geny pro imunoglobuliny genetika   MeSH
• geny TcR genetika   MeSH
• imunoglobuliny genetika   MeSH
• lidé MeSH
• receptory antigenů T-buněk genetika   MeSH
• referenční standardy MeSH
• rekombinace genetická genetika   MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor genetika   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genetické markery MeSH
• imunoglobuliny MeSH
• receptory antigenů T-buněk MeSH

We report a unique case of aleukemic granulocytic sarcoma of the neck, originally misdiagnosed as non-Hodgkin's lymphoma (NHL), though chloroma was also suspected due to a greenish macroscopic appearance and the presence of myeloid chloroacetate esterase (CAE)+ cells. The proof of clonal T cell receptor gamma chain (TcRgamma) gene rearrangements in the recurring tumor was deemed to confirm the initial diagnosis of T cell NHL. Altogether five distinct types of clonal TcRgamma gene rearrangements were found in the tumor, bone marrow and peripheral blood. Only retrospectively, using RT-PCR, did we detect the acute myeloid leukemia subset-specific fusion gene AML1/ETO in the frozen samples of the relapsed tumor, as well as in the otherwise unaffected bone marrow and peripheral blood (representing 'minimal initial disease' in the latter two samples). Simultaneous staining verified that the neoplastic CAE+ cells and CD45RO+ T cells were different populations.

• MeSH
• dospělí MeSH
• fúzní onkogenní proteiny genetika   MeSH
• genová přestavba - gama řetězec receptoru antigenů T-buněk * MeSH
• lidé MeSH
• myeloidní sarkom genetika   imunologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein PEBP2A2 MeSH
• protein RUNX1T1 MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AML1-ETO fusion protein, human MeSH Prohlížeč
• fúzní onkogenní proteiny MeSH
• protein PEBP2A2 MeSH
• protein RUNX1T1 MeSH
• transkripční faktory MeSH

We present a series of 15 cases of cutaneous lymphoma and pseudolymphoma with dual lineage rearrangement identified among approximately 1200 cases of cutaneous lymphoproliferative disorders assessed in our 4 institutions during the last 8 years in which the results of both T-cell receptor and immunoglobulin heavy chain rearrangement investigations were available. On the basis of the clinicopathologic information, the cases were retrospectively subdivided into 2 categories: (1) cases with definite features of cutaneous lymphoma or pseudolymphoma (n = 11) and (2) cases with unclassifiable disease (n = 4). The detection of dual genotype in the first group did not influence the final diagnosis; 7 cases represented cutaneous B-cell lymphomas, 3 pseudolymphomas, and 1 case lymphomatoid papulosis. The presence of monoclonal T-cell receptor-gene rearrangements in these cases may be explained either by monoclonal or oligoclonal expansion of exuberant T cells (or B cells in case of lymphomatoid papulosis) or by lineage infidelity. Three patients with unclassifiable disease had several clinical and histopathologic features in common. They were elderly, presented with solitary lesions, were in good general health and histopathologically demonstrated a dense multinodular infiltrate containing approximately an equal number of T and B cells and a high number of histiocytes forming granulomas, with prominent granulomatous features in 2 cases. B cells were either scattered with the infiltrate or formed collections vaguely resembling follicles; Reed-Sternberg-like cells were seen in 2 cases. B cells showed expression neither of immunoglobulin light chain. The T-cell component was represented mainly by small, well-differentiated lymphocytes or slightly pleomorphic cells, with some medium-sized convoluted cells. Epstein-Barr virus was not detected by polymerase chain reaction. The exact classification of these cases is unknown; they differ histopathologically from previously published cases of bigenotypic cutaneous lymphomas. They may merely represent a growth or reactive pattern, but, on the other hand, may be low-grade lymphomas. If so, they may be histopathologically related to cutaneous Hodgkin disease, T-cell/histiocyte-rich large B-cell lymphoma, or composite lymphomas. Further reports are needed to identify these lesions to clarify their nature and biologic potential.

• MeSH
• B-buněčný lymfom patologie   MeSH
• B-lymfocyty patologie   MeSH
• buňky Reedové-Sternberga patologie   MeSH
• dospělí MeSH
• genová přestavba B-lymfocytů * MeSH
• genová přestavba T-lymfocytů * MeSH
• histiocyty patologie   MeSH
• Hodgkinova nemoc diagnóza   MeSH
• kožní T-buněčný lymfom patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfom patologie   MeSH
• lymfoproliferativní nemoci klasifikace   genetika   patologie   MeSH
• nádory kůže genetika   patologie   MeSH
• polymerázová řetězová reakce MeSH
• pseudolymfom patologie   MeSH
• receptory antigenů T-buněk genetika   MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• T-lymfocyty patologie   MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH
• těžké řetězce imunoglobulinů MeSH

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ ALCL) originates from the T-lineage and is marked by rearrangements of the ALK gene. More than 10 fusion partners with the ALK gene are known, with the most common being the t(2;5)(p23;q35) translocation resulting in the NPM1::ALK fusion. In 10% to 20% of the ALK+ ALCL cases, the ALK gene fuses with various other partners. Modern molecular techniques, especially next-generation sequencing (NGS), have eased the identification of ALK gene fusion partners and have allowed in-depth characterization of the T-cell receptor (TCR) repertoire. We devised a real-time quantitative reverse-transcription polymerase chain reaction to measure the expression of the translocated portion of the ALK gene. Fusion partners for the ALK gene were analyzed using rapid amplification of 5'cDNA ends (RACE) method or NGS. TCR immunoprofiling was performed by amplicon NGS. We studied 96 ALK+ ALCL patients. NPM1::ALK fusion gene was observed in 71 patients, ATIC::ALK in 9, and TPM3::ALK in 3. CLTC::ALK, MYH9::ALK, and RNF213::ALK fusions were identified in 2 patients each. We also discovered the TPM4::ALK and SATB1::ALK fusion genes, plus the following 2 previously unidentified ALK+ ALCL fusions: SQSTM1::ALK and CAPRIN1::ALK. High expression of the translocated ALK gene segment was observed in all 93 analyzed samples. TCR testing was conducted on 23 patients with available DNA. In 18 (78%) patients, we discerned at least one (ranging from 1 to 4) clonal TCR rearrangement. In 59% of the patients, clonal TCR beta junctions corresponded with sequences previously observed in both healthy donors and under various pathological conditions. Reverse-transcriptase quantitative detection of ALK expression is a fast and reliable method for both diagnosing and monitoring treatment response in ALK+ ALCL patients, irrespective of the ALK gene translocation. NGS reveals new ALK translocation partners. Both malignant and reactive TCR repertoires in ALK+ ALCL patients are unique and do not consistently occur among different patients.

• Klíčová slova
• ALK expression, anaplastic large cell lymphoma, fusion genes ALCL, gene/protein ALK, immunoglobulin and T-cell receptor gene rearrangements, next-generation sequencing,
• MeSH
• adenosintrifosfatasy genetika   MeSH
• anaplastická lymfomová kináza genetika   MeSH
• anaplastický velkobuněčný lymfom * genetika   patologie   MeSH
• jaderné proteiny genetika   MeSH
• lidé MeSH
• proteiny buněčného cyklu genetika   MeSH
• receptory antigenů T-buněk genetika   MeSH
• transkripční faktory genetika   MeSH
• translokace genetická MeSH
• tyrosinkinasové receptory genetika   MeSH
• tyrosinkinasy genetika   MeSH
• ubikvitinligasy * MeSH
• vazebné proteiny DNA v oblastech připojení k matrix * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• anaplastická lymfomová kináza MeSH
• CAPRIN1 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• proteiny buněčného cyklu MeSH
• receptory antigenů T-buněk MeSH
• RNF213 protein, human MeSH Prohlížeč
• SATB1 protein, human MeSH Prohlížeč
• transkripční faktory MeSH
• tyrosinkinasové receptory MeSH
• tyrosinkinasy MeSH
• ubikvitinligasy * MeSH
• vazebné proteiny DNA v oblastech připojení k matrix * MeSH

T cell receptor (TCR) genes (TRA/TRD, TRB and TRG) reside in three regions on human chromosomes (14q11.2, 7q34 and 7p14, respectively) and pig chromosomes (7q15.3-q21, 18q11.3-q12 and 9q21-22, respectively). During the maturation of T cells, TCR genes are rearranged by site-specific recombination. Occasionally, interlocus recombination of different TCR genes takes place, resulting in chromosome rearrangements. It has been suggested that the absolute number of these "innocent" trans-rearrangements correlates with the risk of lymphoma. The aims of this work were to assess the frequencies of rearrangements with breakpoints in TCR genes in domestic pig lymphocytes and to compare these with the frequencies of corresponding rearrangements in human lymphocytes by using fluorescence in situ hybridization with chromosome painting probes. We show that frequencies of trans-rearrangements involving TRA/TRD locus in pigs are significantly higher than the frequency of translocations with breakpoints in TRB and TRG genes in pigs and the frequencies of corresponding trans-rearrangements involving TRA/TRD locus in humans. Complex structure of the pig TRA/TRD locus with high number of potential V(D)J rearrangements compared to the human locus may account for the observed differences. Furthermore, we demonstrated that trans-rearrangements involving pig TRA/TRD locus occur at lower frequencies in γδ T cells than in αβ T lymphocytes. The decrease of the frequencies in γδ T cells is probably caused by the absence of TRA recombination during maturation of this T cell lineage. High numbers of innocent trans-rearrangements in pigs may indicate a higher risk of T-cell lymphoma than in humans.

• MeSH
• chromozomy genetika   MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• lymfom T-buněčný genetika   patologie   MeSH
• prasata MeSH
• receptory antigenů T-buněk genetika   MeSH
• rekombinace genetická * MeSH
• translokace genetická * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• receptory antigenů T-buněk MeSH