The early diagnosis of MF and SS may be challenging and is based on a combination of clinical, biological, histological, and molecular criteria. Until now, the search for T-cell clones in skin was mostly performed by standard PCR amplification of the T-cell receptor (TCR) genes and capillary electrophoresis (CE) but false positives and negatives with this technique hamper MF diagnosis.

High throughput sequencing (HTS) of the TCR genes allows the identification and quantification of each T-cell clone through the recognition of CDR3 sequences in tissue samples. It can be used for the sensitive monitoring of the minimal residual disease in CTCL.

However, the interpretation of HTS clonality data remains subjective, as internationally validated thresholds for the definition of a dominant T-cell clone by HTS have not been published. In this study We sought to evaluate the diagnostic performance and define optimal thresholds for T-cell clonality of HTS of TCR beta gene (HTS-TRB) in CTCL diagnosis.

We sought to evaluate the diagnostic performance and define optimal thresholds for T-cell clonality of HTS of TCR beta gene (HTS-TRB) by analyzing the TCF, ratio of productive frequencies (RPF, defined as the ratio between the 1st and 2nd clone frequencies) and clonality score in patients with CTCL compared to inflammatory skin diseases (ID). We retrospectively analyzed HTS-TRB data of lesional skin biopsy samples from 144 patients between 2013 and 2020. CTCL diagnosis was based on international clinical, histopathological and immunohistochemical criteria. Data were analyzed using SPSS software.

A total of 101 samples from patients with certain CTCL, of which 47 MF stage I-IIA, 1 stage IIB, 4 stage III, 35 SS (3), 8 peripheral T-cell lymphomas not otherwise specified (PTCL NOS) or other aggressive epidermotropic CD8-positive T-cell lymphoma and 6 CD30+ lymphoproliferative disorders (3 lymphomatoid papulosis and 3 anaplastic large-cell lymphomas), were collected. In addition, 43 samples from patients with ID were identified.

The performance of the HTS-TRB technique for CTCL diagnosis was analyzed using receiver-operating characteristics (ROC) curves on 101 CTCL and 43 ID cases. The TCF showed the highest diagnostic performance with an area under curve of 0.957 versus 0.929 for RPF.

With 5% and 25% TCF thresholds, the specificity for CTCL diagnosis was 95% and 100%, and sensitivity 89% and 50%, respectively. Such a high specificity allows to early identify CTCL in difficult cases.

Further multicentre prospective studies are needed to validate international criteria for T-cell clonality analysis by HTS of TCR genes in the diagnosis of CTCL.