TCRbeta Gene Rearrangements for Detection and Monitoring of Minimal Residual Disease after Allogenic Stem-Cell Transplantation in Patients with Advanced Mycosis Fungoides and Sézary Syndrome.
Authors: Corti Laura , Saporiti Giorgia , Fermo Elisa , Berti Emilio , Venegoni Luigia , Deliliers Giorgio Lambertenghi , Onida Francesco 
Published: April 2020

Abstract
Advanced tumor-stage mycosis fungoides (MF) and Sézary syndrome (SS) are cutaneous T-cell lymphomas (CTCL) characterized by very poor prognosis. Allogeneic haematopoietic stem cell transplantation (allo-SCT) represents an experimental treatment which has been shown to be very effective in achieving long lasting complete remission, possibly leading to cure in selected patients. However, high transplant-related mortality (TRM) limit its feasibility in the vast majority of patients with CTCL. Reduced-intensity conditioning regimens have been demonstrated to decrease TRM, allowing gradual establishment of full donor chimerism and possible graft-versus-lymphoma effect. In this setting, evaluation of minimal residual disease (MRD) is particularly useful to guide post-transplant strategies such as donor lymphocyte infusion (DLI). Detection of TCRgamma-chain gene rearrangements, owing to the relatively limited complexity of its genetic elements, is routinely used to detect MRD in T-cell malignancies. Nonetheless, due to the extensive combinatorial repertoire and the large hypervariable regions, TCRbeta represents the best target for MRD monitoring, allowing more sensitive detection of patient-specific rearrangements. With this study, we aimed to identify patient-specific TCRbeta rearrangements to monitor MRD in patients enrolled in a clinical phase II trial of reduced intensity allo-SCT for advanced stage refractory MF/SS. Skin biopsy and peripheral blood samples at diagnosis and at different time points after transplant were obtained from 6 out of 9 evaluable patients, all having achieved clinical complete remission (still enduring in 5). The BIOMED-2 multiplex TCRbeta PCR heteroduplex assay (InVivoScribe Technologies, USA) was used for identification of monoclonal TCRbeta rearrangements, clonal PCR products were directly sequenced in both directions using the complete set of V or J primers, and V, D, J segments identified using the ImMunoGeneTics database (http://imgt.cines.fr). Then, clonespecific PCR assays were performed on samples collected from every single patient and specificity tested by parallel amplification of normal polyclonal DNA samples. In all patients a monoclonal TCRbeta rearrangement has been sequenced allowing to obtain clone-specific primers for PCR assays. In 2 patients we identified the presence of a MRD at the early controls after transplant (+2 and +3 months, respectively) when both were polyclonal by standard TCRgamma rearrangement; clone-specific PCR assays for TCRbeta became negative afterwards, in concomitance with the achievement of full donor chimerism. In 3 patients polyclonality of TCRbeta was observed in all post-transplant controls. One developed chronic cutaneous GvHD and skin biopsies verified the absence of clone-specific T-lymphocytes. In another patient, all post-transplant assays performed by the standard TCRgamma-rearrangement were persistently positive, suggesting the presence of a non disease-specific monoclonality. The last of the 6 patients relapsed 52 months after transplant. Retrospective clone-specific analysis of TCRbeta rearrangements in DNA from skin biopsies unveiled the presence of molecular relapse already 24 months before, whereas standard TCRgamma assays became positive only after clinical relapse. Altogether, we observed good stability of monoclonal TCRbeta rearrangements over time. Our results suggest that the analysis of TCRbeta is more sensitive and more specific than the analysis of standard TCRgamma for detection of MRD in CTCLs, allowing earlier identification of relapses and adopatiention of pre-empatientive treatment such as DLI. With this method, we also demonstrated disappearance of MRD in concomitance with the achievement of full donor chimerism after allo-SCT. Finally, disease clone-specific TCRbeta rearrangement detection might be helpful in distinguishing cutaneous manifestations of acute GvHD from early relapse of MF. TCRbeta analyses of samples from the remaining 3 evaluable patients are ongoing and will be included in the final presentation.