Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma
Analysis of T-cell receptor (TCR) clonality is a major diagnostic tool for lymphomas, particularly for cutaneous T-cell lymphomas (CTCL) like Mycosis fungoides and Sézary syndrome. However, a fast and cost-effective workflow is needed to enable widespread use of this method. : We established a proce...
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| Veröffentlicht in: | Cancers 2024-11, Vol.16 (21), p.3700 |
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| creator | Cieslak, Cassandra Hain, Carsten Rückert-Reed, Christian Busche, Tobias Klages, Levin Joe Schaper-Gerhardt, Katrin Gutzmer, Ralf Kalinowski, Jörn Stadler, Rudolf |
| description | Analysis of T-cell receptor (TCR) clonality is a major diagnostic tool for lymphomas, particularly for cutaneous T-cell lymphomas (CTCL) like Mycosis fungoides and Sézary syndrome. However, a fast and cost-effective workflow is needed to enable widespread use of this method.
: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison.
The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992-0.996; range r
: 0.984-0.991).
: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use. |
| doi_str_mv | 10.3390/cancers16213700 |
| format | Article |
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: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison.
The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992-0.996; range r
: 0.984-0.991).
: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use.</description><identifier>ISSN: 2072-6694</identifier><identifier>EISSN: 2072-6694</identifier><identifier>DOI: 10.3390/cancers16213700</identifier><identifier>PMID: 39518138</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Accuracy ; Bar codes ; Biopsy ; Cloning ; Costs ; Development and progression ; DNA sequencing ; Fungal infections ; Instrument industry ; International economic relations ; Lymphocytes T ; Lymphoma ; Medical prognosis ; Mycoses ; Mycosis ; Mycosis fungoides ; Next-generation sequencing ; Non-Hodgkin's lymphomas ; Nucleotide sequencing ; Patients ; Skin ; T cell receptors ; T cells ; T-cell lymphoma ; Working groups</subject><ispartof>Cancers, 2024-11, Vol.16 (21), p.3700</ispartof><rights>COPYRIGHT 2024 MDPI AG</rights><rights>2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2024 by the authors. 2024</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c332t-13cf364d7edbeea188865b098318710c67d0abaff5b7ce0a780a17feb3d57bd33</cites><orcidid>0000-0002-9722-4435 ; 0000-0001-9211-8927 ; 0000-0003-2683-6028 ; 0000-0002-9052-1998 ; 0000-0001-7921-2820</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC11544856/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC11544856/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/39518138$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Cieslak, Cassandra</creatorcontrib><creatorcontrib>Hain, Carsten</creatorcontrib><creatorcontrib>Rückert-Reed, Christian</creatorcontrib><creatorcontrib>Busche, Tobias</creatorcontrib><creatorcontrib>Klages, Levin Joe</creatorcontrib><creatorcontrib>Schaper-Gerhardt, Katrin</creatorcontrib><creatorcontrib>Gutzmer, Ralf</creatorcontrib><creatorcontrib>Kalinowski, Jörn</creatorcontrib><creatorcontrib>Stadler, Rudolf</creatorcontrib><title>Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma</title><title>Cancers</title><addtitle>Cancers (Basel)</addtitle><description>Analysis of T-cell receptor (TCR) clonality is a major diagnostic tool for lymphomas, particularly for cutaneous T-cell lymphomas (CTCL) like Mycosis fungoides and Sézary syndrome. However, a fast and cost-effective workflow is needed to enable widespread use of this method.
: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison.
The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992-0.996; range r
: 0.984-0.991).
: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use.</description><subject>Accuracy</subject><subject>Bar codes</subject><subject>Biopsy</subject><subject>Cloning</subject><subject>Costs</subject><subject>Development and progression</subject><subject>DNA sequencing</subject><subject>Fungal infections</subject><subject>Instrument industry</subject><subject>International economic relations</subject><subject>Lymphocytes T</subject><subject>Lymphoma</subject><subject>Medical prognosis</subject><subject>Mycoses</subject><subject>Mycosis</subject><subject>Mycosis fungoides</subject><subject>Next-generation sequencing</subject><subject>Non-Hodgkin's lymphomas</subject><subject>Nucleotide sequencing</subject><subject>Patients</subject><subject>Skin</subject><subject>T cell receptors</subject><subject>T cells</subject><subject>T-cell lymphoma</subject><subject>Working groups</subject><issn>2072-6694</issn><issn>2072-6694</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNptkkFv3CAQhVHVqonSnHurLPXSixPw2IBP1WrVtJVWrZSkpx4QxsOGyAYX7Er770O6SZpEgQMDfO_BjIaQ94yeALT01GhvMCbGKwaC0lfksKKiKjlv69eP4gNynNI1zQOACS7ekgNoGyYZyEPy-4f2YQoRiwv8s6A3zm8LG2JxWa5xGIpzNDjNeX-OOkbttziin4uV18MuuVQ4X6yXWXsMS7rXbHbjdBVG_Y68sXpIeHy3HpFfZ18u19_Kzc-v39erTWkAqrlkYCzwuhfYd4iaSSl509FWApOCUcNFT3WnrW06YZBqIalmwmIHfSO6HuCIfN77Tks3Ym_yB6Me1BTdqONOBe3U0xvvrtQ2_FWMNXUtG54dPt05xJCrkGY1umRyLvvEFLBKihqEaDP68Rl6HZaYy_GPatqWA63-U1s9oHLehvywuTVVK8kaqOuaykydvEDl2ePoTPBoXT5_IjjdC0wMKUW0D0kyqm57Qj3riaz48Lg2D_x9B8AN9C2yzQ</recordid><startdate>20241101</startdate><enddate>20241101</enddate><creator>Cieslak, Cassandra</creator><creator>Hain, Carsten</creator><creator>Rückert-Reed, Christian</creator><creator>Busche, Tobias</creator><creator>Klages, Levin Joe</creator><creator>Schaper-Gerhardt, Katrin</creator><creator>Gutzmer, Ralf</creator><creator>Kalinowski, Jörn</creator><creator>Stadler, Rudolf</creator><general>MDPI AG</general><general>MDPI</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7T5</scope><scope>7TO</scope><scope>7XB</scope><scope>8FE</scope><scope>8FH</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>H94</scope><scope>HCIFZ</scope><scope>LK8</scope><scope>M2O</scope><scope>M7P</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-9722-4435</orcidid><orcidid>https://orcid.org/0000-0001-9211-8927</orcidid><orcidid>https://orcid.org/0000-0003-2683-6028</orcidid><orcidid>https://orcid.org/0000-0002-9052-1998</orcidid><orcidid>https://orcid.org/0000-0001-7921-2820</orcidid></search><sort><creationdate>20241101</creationdate><title>Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma</title><author>Cieslak, Cassandra ; 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However, a fast and cost-effective workflow is needed to enable widespread use of this method.
: We established a procedure for TCR rearrangement analysis via Oxford Nanopore Technology (ONT) sequencing. TCR receptor rearrangements (TCR-gamma and TCR-beta chains) were analyzed in samples from 45 patients with various diagnoses: Mycosis fungoides (37/45), Sézary Syndrome (2/45), folliculotropic CTCL (1/45), and non-CTCL diagnoses as polyclonal controls (5/45). Sample types included formalin-fixed paraffin-embedded (FFPE) samples (27/45), fresh frozen samples (9/45), and CD3-isolated cells (9/45). In addition, DNA of a Jurkat cell line was used as a monoclonal control. TCR amplicons were generated employing an optimized version of the protocol from the Euro Clonality consortium. Sequencing was conducted on the ONT GridION and Illumina MiSeq platforms, followed by similar bioinformatic analysis protocols. The tumor clone frequency (TCF), a crucial prognostic factor for CTCL patients, was used for method comparison.
The use of an optimized amplicon protocol and adapted bioinformatic tools demonstrated a strong correlation in TCF values between both sequencing methods across all sample types (range R: 0.992-0.996; range r
: 0.984-0.991).
: In summary, ONT sequencing was able to detect TCR clonality comparable to NGS, indicating its potential as a faster and more cost-effective option for routine diagnostic use.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>39518138</pmid><doi>10.3390/cancers16213700</doi><orcidid>https://orcid.org/0000-0002-9722-4435</orcidid><orcidid>https://orcid.org/0000-0001-9211-8927</orcidid><orcidid>https://orcid.org/0000-0003-2683-6028</orcidid><orcidid>https://orcid.org/0000-0002-9052-1998</orcidid><orcidid>https://orcid.org/0000-0001-7921-2820</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Accuracy Bar codes Biopsy Cloning Costs Development and progression DNA sequencing Fungal infections Instrument industry International economic relations Lymphocytes T Lymphoma Medical prognosis Mycoses Mycosis Mycosis fungoides Next-generation sequencing Non-Hodgkin's lymphomas Nucleotide sequencing Patients Skin T cell receptors T cells T-cell lymphoma Working groups |
| title | Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma |
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