Radiotherapy Eradicates Malignant T Cells and Is Associated with Improved Survival in Early-Stage Mycosis Fungoides.

PURPOSE: Mycosis fungoides is the most common subtype of cutaneous T-cell lymphoma. Skin-directed treatments often improve but do not cure mycosis fungoides skin lesions. The purpose of this study was to (i) assess whether remission was associated with malignant T-cell clone depletion at treated sites using either low-dose radiotherapy (LDRT, 8 Gy) or topical steroids and (ii) assess whether a clone-ablative therapy, like LDRT, is associated with overall survival in patients with high-risk early-stage CTCL. EXPERIMENTAL DESIGN: Pre- and posttreatment biopsies from 20 lesional skin samples of 18 patients with mycosis fungoides who received either 8 Gy LDRT (n = 16) or topical steroids (n = 4) underwent high-throughput T-cell receptor sequencing of the TCRB gene to quantify the malignant T-cell clone. For the retrospective chart review, overall survival of 47 high-risk early-stage patients was compared between patients who did or did not receive radiation. RESULTS: LDRT eradicated the clone in 5 of 16 lesions and reduced it >90% in 11 of 16; there were no recurrences in these lesions. Patients treated with topical steroids appeared to clinically improve, but the malignant clone persisted. We found that the number of residual malignant T cells predicted lesion recurrence. A retrospective review showed that early-stage high-risk patients who received radiation as part of their treatment regimen had prolonged overall survival compared with patients who did not. CONCLUSIONS: These findings demonstrate that LDRT can eradicate malignant T cells in mycosis fungoides, provides robust disease control, and is associated with improved survival in high-risk early-stage patients.

Benign T cells drive clinical skin inflammation in cutaneous T cell lymphoma.

Psoralen plus UVA (PUVA) is an effective therapy for mycosis fungoides (MF), the skin-limited variant of cutaneous T cell lymphoma (CTCL). In low-burden patients, PUVA reduced or eradicated malignant T cells and induced clonal expansion of CD8+ T cells associated with malignant T cell depletion. High-burden patients appeared to clinically improve but large numbers of malignant T cells persisted in skin. Clinical improvement was linked to turnover of benign T cell clones but not to malignant T cell reduction. Benign T cells were associated with the Th2-recruiting chemokine CCL18 before therapy and with the Th1-recruiting chemokines CXCL9, CXCL10, and CXCL11 after therapy, suggesting a switch from Th2 to Th1. Inflammation was correlated with OX40L and CD40L gene expression; immunostaining localized these receptors to CCL18-expressing c-Kit+ dendritic cells that clustered together with CD40+OX40+ benign and CD40+CD40L+ malignant T cells, creating a proinflammatory synapse in skin. Our data suggest that visible inflammation in CTCL results from the recruitment and activation of benign T cells by c-Kit+OX40L+CD40L+ dendritic cells and that this activation may provide tumorigenic signals. Targeting c-Kit, OX40, and CD40 signaling may be novel therapeutic avenues for the treatment of MF.

High-throughput sequencing of the T cell receptor ß gene identifies aggressive early-stage mycosis fungoides.

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor ß gene (TCRB) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

Clinically resolved psoriatic lesions contain psoriasis-specific IL-17-producing αß T cell clones.

In psoriasis, an IL-17-mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular Vß and Vα subfamilies. We identified 15 TCRß and 4 TCRα antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of αß versus γδ T cells in psoriasis, we carried out TCR/δ HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are αß T cells. γδ T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were αß T cells. In summary, IL-17-producing αß T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.

TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.

Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin.