Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Nov 1;16(21):3700.
doi: 10.3390/cancers16213700.

Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma

Cassandra Cieslak  1 , Carsten Hain  2 , Christian Rückert-Reed  2 , Tobias Busche  2 , Levin Joe Klages  2 , Katrin Schaper-Gerhardt  1 , Ralf Gutzmer  1 , Jörn Kalinowski  2 , Rudolf Stadler  1
Affiliations

Nanopore Sequencing for T-Cell Receptor Rearrangement Analysis in Cutaneous T-Cell Lymphoma

Cassandra Cieslak et al. Cancers (Basel). .

Abstract

Background: 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. Methods: 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. Results: 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 r2: 0.984-0.991). Conclusions: 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.

Keywords: Mycosis fungoides; T-cell receptor rearrangement analysis; cutaneous T-cell lymphoma; nanopore sequencing.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. R.G.: Research support: Amgen, MerckSerono, SUN Pharma, Sanofi/Regeneron, Admiral Hermal, Kyowa Kirin. Honoraria for lectures: Bristol-MyersSquibb, Novartis, MSD, Almirall-Hermal, Amgen, Merck-Serono, SUN, Pierre-Fabre, Sanofi/Regeneron, SUN Pharma Honoraria for advisory boards: Bristol-MyersSquibb, Novartis, MSD, Almirall-Hermal, Amgen, Pierre-Fabre, Merck-Serono, 4SC, Immunocore, SUN Pharma, Sanofi/Regeneron, Delcath. RS: Kyowa Kirin, 4SC Ag, Stemline, Innate Pharma.

Figures

Figure 1
Figure 1
Correlation of TCF data for TCR analysis via Illumina (MiSeq) or ONT amplicon sequences. For all samples the calculated TCF values for both analyzed T-cell receptor chains (TRG and TRB) are shown and correlation between both sequencing methods is depicted. (a,b) FFPE samples, TRG n = 27, TRB n = 23. (c,d) CD3-isolated cell samples, TRG n = 9, TRB n = 9. (e,f) fresh frozen (FF) samples, TRG n = 9, TRB n = 9.
Figure 2
Figure 2
Chord diagrams of TRG repertoires. A monoclonal sample in TRG sequenced by MiSeq (a) and ONT (b) compared to a polyclonal example in TRG analyzed with MiSeq (c) and ONT (d).
Figure 3
Figure 3
Relative quantification of the top clones (TCF) in replicates for 3 samples: a healthy donor, Jurkat cell line and a PBMC sample isolated from a patient with active immune state. The blue spots are the MiSeq (Illumina) data; the green spots, the ONT-Q20 data (Guppy Version 5.1.13); and the one spot in purple (in TRG–Jurkat 100%) is another ONT-Q20 spot analyzed with Guppy version 6.1.5.
See this image and copyright information in PMC

References

    1. Hodak E., Amitay-Laish I., Atzmony L., Prag-Naveh H., Yanichkin N., Barzilai A., Kershenovich R., Feinmesser M. New Insights into Folliculotropic Mycosis Fungoides (FMF): A Single-Center Experience. J. Am. Acad. Dermatol. 2016;75:347–355. doi: 10.1016/j.jaad.2016.03.009. - DOI - PubMed
    1. Tomasini C., Kempf W., Novelli M., Fava P., Annessi G., Rongioletti F., Fierro M.T., Quaglino P. Spiky Follicular Mycosis Fungoides: A Clinicopathologic Study of 8 Cases. J. Cutan. Pathol. 2015;42:164–172. doi: 10.1111/cup.12399. - DOI - PubMed
    1. van Santen S., Roach R.E.J., van Doorn R., Horváth B., Bruijn M.S., Sanders C.J.G., de Pooter J.C., van Rossum M.M., de Haas E.R.M., Veraart J.C.J.M., et al. Clinical Staging and Prognostic Factors in Folliculotropic Mycosis Fungoides. JAMA Dermatol. 2016;152:992–1000. doi: 10.1001/jamadermatol.2016.1597. - DOI - PubMed
    1. Treatment of Early-Stage Mycosis Fungoides: Results from the PROspective Cutaneous Lymphoma International Prognostic Index (PROCLIPI) Study. [(accessed on 10 January 2024)]; Available online: https://pubmed.ncbi.nlm.nih.gov/32479678/ - PMC - PubMed
    1. Scarisbrick J.J., Kim Y.H., Whittaker S.J., Wood G.S., Vermeer M.H., Prince H.M., Quaglino P. Prognostic Factors, Prognostic Indices and Staging in Mycosis Fungoides and Sézary Syndrome: Where Are We Now? Br. J. Dermatol. 2014;170:1226–1236. doi: 10.1111/bjd.12909. - DOI - PubMed

LinkOut - more resources