Topical resiquimod can induce disease regression and enhance T-cell effector functions in cutaneous T-cell lymphoma

Abstract

Early-stage cutaneous T-cell lymphoma (CTCL) is a skin-limited lymphoma with no cure aside from stem cell transplantation. Twelve patients with stage IA-IIA CTCL were treated in a phase 1 trial of 0.03% and 0.06% topical resiquimod gel, a Toll-like receptor 7/8 agonist. Treated lesions significantly improved in 75% of patients and 30% had clearing of all treated lesions. Resiquimod also induced regression of untreated lesions. Ninety-two percent of patients had more than a 50% improvement in body surface area involvement by the modified Severity-Weighted Assessment Tool analysis and 2 patients experienced complete clearing of disease. Four of 5 patients with folliculotropic disease also improved significantly. Adverse effects were minor and largely skin limited. T-cell receptor sequencing and flow cytometry studies of T cells from treated lesions demonstrated decreased clonal malignant T cells in 90% of patients and complete eradication of malignant T cells in 30%. High responses were associated with recruitment and expansion of benign T-cell clones in treated skin, increased skin T-cell effector functions, and a trend toward increased natural killer cell functions. In patients with complete or near eradication of malignant T cells, residual clinical inflammation was associated with cytokine production by benign T cells. Fifty percent of patients had increased activation of circulating dendritic cells, consistent with a systemic response to therapy. In summary, topical resiquimod is safe and effective in early-stage CTCL and the first topical therapy to our knowledge that can induce clearance of untreated lesions and complete remissions in some patients. This trial was registered at www.clinicaltrials.gov as #NCT813320.

Trial registration: ClinicalTrials.gov NCT01676831.

© 2015 by The American Society of Hematology.

Figures

Figure 1

Topical resiquimod therapy can induce the regression of both treated and untreated skin lesions. (A) Representative clinical images are shown of treated CTCL skin lesions before and after treatment in 3 patients. Lesions are shown before therapy (top) and at the indicated times after institution of therapy. (B) Representative clinical images of an untreated CTCL lesion before therapy (top) and at 16 weeks after beginning therapy to other sites (bottom).

Figure 2

Resiquimod is an effective therapy for early-stage CTCL. Changes from baseline of CAILS (A) and SWAT scores (B) are shown for the 12 patients treated. Patient numbers are shown below each bar and the dosage of topical resiquimod is indicated by bar color. Nine of 12 patients had a 50% or greater improvement from baseline of treated lesions among and 4 cleared all treated target lesions during therapy. Eleven of 12 patients experienced improvement of 50% or more in total surface area of involvement from baseline as assessed by SWAT score analysis, with 2 patients experiencing complete clearing of all evidence of disease. CR, complete remission; PR, partial remission; SD, stable disease.

Figure 3

Evaluation of benign and malignant T cells in skin lesions before and after resiquimod therapy using HTS TCR analysis. DNA was isolated from lesional skin before and after resiquimod therapy and analyzed by HTS TCR-β analysis. HTS results before (A,C) and after (B,D) resiquimod therapy for 2 patients with a complete response are shown. This technique allows identification and quantification of the malignant T-cell clone as well as measurement of the exact number, diversity, relative proportions, and sequences of all T cells in the sample. (E,F) Clinical images of a treated lesion from patient 11 are shown. V, variable.

Figure 4

Nine of 10 patients tested had reduction in the frequency of malignant T cells in skin following resiquimod therapy. HTS of DNA from lesional skin before therapy and at 8 weeks was used to identify and quantify clonal malignant T cells. (A) The change in frequency of the malignant T-cell clone as a percentage of the total T-cell population is shown. Patients with a clinical CR (patients 3 and 8) and PR (all others) are indicated. Patient 11, a clinical PR, had eradication of the malignant clone at 8 weeks and patient 1, a clinical PR, had a 99.6% reduction in the malignant clone. Only patient 6 showed a slight increase in the malignant T-cell frequency. (B) The correlation of clinical SWAT scores with malignant T-cell clone frequency by HTS are shown. In general, clinical improvement lagged behind clearance of the malignant T-cell clone and in a subset of patients (11, 1, 5), clinical inflammation persisted despite complete or near eradication of the malignant clone.

Figure 5

Eradication of malignant T cells correlated with recruitment and expansion of new responding benign T cells, but was not related to the initial burden of malignant clonal T cells or the total number of benign T cells before or after therapy. (A) The absolute numbers of malignant T cells per 100 µg of DNA before (pre-tx) and after therapy (post-tx) are shown. There was no correlation of a complete or near-complete eradication of malignant T cells (patients 3, 8,1 1, and 1) with initial clonal T-cell burden. (B) The absolute number of benign infiltrating T cells before and after therapy are shown and did not correlate with clearance of the malignant T-cell clone. (C) Expansion of benign T-cell clones in treated lesions was associated with improved clinical responses. The number of expanded (>1% frequency) benign infiltrating T-cell clones in patients after treatment are shown and was associated with improved clinical responses and eradication of clonal malignant T cells. (D) The percentage of the top 20 benign T-cell clones that were newly recruited vs resident in the skin before treatment are shown. The 20 most frequent benign T-cell clones were identified after therapy and their presence before treatment was determined. Patients were divided into high responders (high resp; >99% malignant T-cell eradication), mid-responders (mid resp; 50% to 99% eradication), and low responders (low resp; 0% to 50% eradication). Recruitment of new responding benign T-cell clones into treated lesions was associated with better eradication of malignant T cells. (E-F) The frequency and absolute numbers of the top 10 most frequent benign T-cell clones pre-tx and post-tx are shown in examples of high-responding (HR) and low-responding (LR) patients. In general, HR patients had marked expansion of benign T-cell clones. Pt, patient.

Figure 6

Malignant T-cell eradication is associated with increased T-cell and NK-effector functions in treated skin. NK and T cells were extracted from lesional skin before and at 8 weeks after resiquimod therapy in 3 HR (1, 3, and 8) and 3 LR (4-6) patients and production of cytokines and effector molecules was evaluated by intracellular cytokine staining and flow cytometry analysis after PMA and ionomycin stimulation. (A-C) Malignant clonal T-cell eradication was associated with high levels of IFN-γ production by both CD4+ and CD8+ T cells. (A) IFN-γ and IL-17 production by CD4+ T cells from patient 3, who had eradication of malignant T cells and a complete clinical response, is shown. Individual patients, before and after resiquimod therapy (B) and aggregate measurements after resiquimod therapy (C) of T-cell IFN-γ production are shown. CD4+ T-cell production of IFN-γ was significantly higher in HR patients. (D) Production of TNF-α, perforin, and granzyme by CD4+ and CD8+ T cells after therapy is shown. Production of TNF-α by CD4+ T cells and granzyme by CD8+ T cells was significantly higher in HR patients. (E) Increased NK effector functions were associated with clearance of the malignant clone. Production of IFN-γ, perforin, and granzyme by CD56+ NK cells is shown. There was a trend for increased NK effector functions in HR patients but given the low number of samples, these differences were not significant. *P < .05, **P < .01.

Figure 7

TCR sequencing is superior to clinical score evaluation in assessing clearance of the malignant T-cell clones and resiquimod is associated with maturation of circulating DCs. (A) Although in many patients, clinical scores improved following reduction of the malignant T cell clone, at least 3 patients (1, 5, and 11) still had clinically evident disease despite near-complete eradication of malignant T cells. The percentage malignant clone and clinical CAILS scores for patients 1 and 5 are shown. (B) Likewise, improvement in clinical score did not necessarily reflect depletion of malignant T cells. Results for patients 6 are shown, in whom the percentage and absolute number of malignant T cells increased despite a clinically improving examination. (C) Residual inflammation on clinical examination is more reflective of benign T-cell activation than the presence of malignant T cells. The CAIL clinical scores at 12 weeks, the percentage of benign T and NK cells making the indicated cytokines, and the percentage of malignant clonal T cells are shown together for patients 1, 5, and 6. In patients 1 and 5, activation of benign T cells was evident despite near-complete eradication of the malignant T-cell clone, suggesting benign T cells were producing the inflammation measured by clinical CAILS scores. In patient 6, low cytokine production by benign T cells was associated with an improving clinical score but an increasing burden of malignant T cells. (D) CD80 expression is increased on circulating CD11c+ myeloid-derived DCs during periods of topical resiquimod use. PBMCs were isolated from peripheral blood at the indicated time points and expression of CD80 on CD11c+ DC was assessed by flow cytometry. Periods when patients were applying resiquimod gel are indicated. Representative results from 2 patients are shown; a similar pattern of DC maturation was observed in 4/8 patients tested. (E) Resiquimod further enhanced DC maturation in vitro. PBMCs isolated from blood at the indicated time points were incubated overnight in 10 µg/mL resiquimod and surface expression of CD80 on CD11c+ DCs was assessed by flow cytometry.