Tumor infiltrating lymphocytes as adjuvant treatment in stage III melanoma patients with only one invaded lymph node after complete resection: results from a multicentre, randomized clinical phase III trial.

BACKGROUND: Adoptive tumor-infiltrating lymphocytes (TIL) therapy and interleukin-2 (IL-2) have been investigated in melanoma. AIM: To confirm previously observed preventive effects of TIL + IL2 in a subgroup of patients with relapsing metastatic stage III melanoma. METHODOLOGY: Open-label, randomized two-group, multicenter five-year trial in adult stage III melanoma patients with only one invaded lymph node after complete resection. Patients received TIL + IL2 or abstention. TIL + IL2 was administered within 8 weeks after lymph node resection and 4 weeks after. Disease-free survival was assessed every 2 months up to month 18, every 3 months up to month 36 and every 4 months up to 5 years. A once-a-year follow-up was scheduled beyond the five-year follow-up. Safety was assessed throughout the trial. RESULTS: Overall, 49 patients accounted for the modified intent-to-treat and 47 for the PP. Slightly more male than female patients participated; mean age was 57.7 ± 11.4 years in the TIL + IL2 group and 53.5 ± 13.0 years in the abstention group. After 5 years of follow-up, 11/26 patients in the TIL + IL2 group and 13/23 in the abstention group had relapsed. There was no statistical difference between the groups (HR: 0.63 CI 95% [0.28-1.41], p = 0.258), nine patients in the TIL + IL2 and 11 in the abstention group died with no significant difference between the two groups (HR: 0.65 CI95% [0.27 - 1.59], p = 0.34). Safety was good. CONCLUSION: We did not confirm results of a previous trial. However, ulceration of the primary melanoma may be considered predictive of the efficacy of TIL in melanoma in adjuvant setting, in a manner similar to interferon α.

BRAF mutations might be more common than supposed in vulvar melanomas.

Data on BRAF, NRAS and KIT mutations are scarce in patients with vulvo-vaginal melanomas and are associated with important therapeutic issues. We investigated their prevalence in a cohort of patients with female lower genital tract melanomas between 2003 and 2017. Of the 22 patients, 5 (22.7%) harboured a BRAF mutation, which was much higher than the rate of 5% reported in the literature. One patient, who was tested negative on the primary melanoma, had a NRAS mutation in a cutaneous metastasis. Our data provide a rationale for prospective and repeated mutations testing in female lower genital tract melanomas.

Clinical, Genetic and Innate Immunity Characteristics of Melanoma in Organ Transplant Recipients.

The aims of this study were to determine the clinical and histological characteristics of melanoma in transplant recipients, the mutation profile (BRAF, NRAS and c-KIT genes), and the immune tolerance of the tumour microenvironment by immunohistochemical study of the expression of indoleamine 2,3-dioxygenase (IDO), PD1, PD-L1, CD8 and FoxP3. The study population comprised patients who had undergone a renal transplant in Nantes University Hospital who developed post-transplantation melanoma. Twenty cases of melanoma out of 4,663 transplant recipients were studied. The results differed from the usual data with respect to melanoma site: 40% were located on the face and were of the malignant lentigo type. The mutation profile was concordant with that of the immunocompetent population. IDO was expressed in all the sections tested, while CD8, FoxP3, PD1 and PD-L1 were poorly expressed. This reflected a highly immunodepressed tumour environment, raising the question of the inductive role of IDO on tumour immune tolerance in patients presenting with long-term immunodepression.

Adoptive T cell therapy combined with intralesional administrations of TG1042 (adenovirus expressing interferon-γ) in metastatic melanoma patients.

Tumor immune escape has recently been shown to be related to the development of an immune tolerance state of the microenvironment. Cytokines activating the immune system such as IFN-γ can be used to reverse the immune escape and thus to potentiate the efficacy of immunotherapy. A clinical study was conducted in 18 stage IIIc/IV melanoma patients treated with tumor-infiltrating lymphocytes (TILs) in combination with intratumoral TG1042 injection (adenovirus expressing IFN-γ). The primary objective was to investigate the safety of treatment. Secondary objectives were to study the clinical response and translational research. The treatment was well tolerated. Among the 13 patients evaluable for tumor response, 38.5% had an overall objective response (OOR = CR + PR) and disease control rate (DCR = CR + PR + S) of 46%. The clinical response of the 37 targeted lesions led to an OOR of 51% and a DCR of 75%. Translational research on predictive markers did not significantly differ between responder and non-responder patients. However, specifically regarding injected lesions, the clinical response correlated with CD3-/CD56+ NK cells which could be activated by TG1042. Further larger studies of this combined immunotherapy are needed to confirm our findings. Intralesional TG1042 combined with antigen-selected TILs should be discussed.

Comparative analysis of BRAF, NRAS and c-KIT mutation status between tumor tissues and autologous tumor cell-lines of stage III/IV melanoma.

In the last decade, advances in molecular biology have provided evidence of the genotypic heterogeneity of melanoma. We analysed BRAF, NRAS and c-KIT alterations in tissue samples from 63 stage III/IV melanoma patients and autologous cell-lines, using either allele-specific or quantitative PCR. The expression of BRAF V600E protein was also investigated using an anti-BRAF antibody in the same tissue samples. 81% of FFPE samples and tumor cell-lines harboured a genetic alteration in either BRAF (54%) or NRAS (27%) oncogenes. There was a strong concordance (100%) between tissue samples and tumor cell-lines. The BRAF V600E mutant-specific antibody showed high sensitivity (96%) and specificity (100%) for detecting the presence of a BRAF V600E mutation. The correlation was of 98% between PCR and immunohistochemistry results for BRAF mutation. These results suggest that BRAF and NRAS mutation status of tumor cells is not affected by culture conditions.

Vorinostat for refractory or relapsing epidermotropic T-cell lymphoma: a retrospective cohort study of 15 patients.

Since the approval of vorinostat for the treatment of refractory cutaneous epidermotropic T-cell lymphoma (CTCL) in 2006, very little data about this treatment have been published. The aim of this retrospective study was to assess the efficacy and safety of vorinostat in patients with CTCL treated between 2007 and 2013 in our department. Fifteen patients (median age 64 years) were included: 9 with Sézary syndrome and 6 with mycosis fungoides. They were all in progression and the median number of systemic treatments previously administered was 3 (range 1-7). With vorinostat treatment, the best response was partial remission in 5 patients (33%) and stabilization in 4 patients (27%). Six patients experienced disease progression. The mean time to response and response duration were 70 (range 31-140) and 300 days (range 157-663), respectively. The most frequent adverse events were asthenia, weight loss, nausea and anaemia. Vorinostat could be a therapeutic alternative for CTCL after treatment failure.

[Melanoma: Cellular and vaccinal immunotherapy]. / Melanome - Thérapeutique immunitaire: l'immunothérapie cellulaire et vaccinale.

Melanoma is a malignancy in which the immune system plays a central role, thus explaining the effectiveness of therapeutic vaccination and cellular immunotherapy with tumor-infiltrating lymphocytes. The identification of specific melanoma antigens was an important step in the development of these new approaches. These treatments are capable of yielding tumor responses that last several years, but the response rate is currently inadequate. The crucial role of the tumor microenvironment has recently been shown: melanoma cells render their immediate environment immunotolerant, undermining the effectiveness of stimulated cytotoxic T lymphocytes. The mechanisms responsible for this state of immune tolerance are a major focus of research. Current therapeutic strategies are based on early adjuvant approaches, destruction of regulatory T cells by lymphodepletion prior to immunotherapy, selection of the melanoma antigens inducing the best cytotoxic T cell responses, and combining cellular therapy with monoclonal antibodies that block molecules inhibiting T lymphocyte activation. Immune therapy for melanoma is thus moving towards adjuvant strategies for early-stage disease and combined treatments for metastatic melanoma. It is also important to identify markers that can be used to predict which patients will respond to a given treatment.

Different strains of Propionibacterium acnes modulate differently the cutaneous innate immunity.

Acne is a chronic inflammatory illness of the pilosebaceous follicle where innate immunity plays a central role. In acne, the density of Propionibacterium acnes is increased in the pilosebaceous unit. We hypothesized that the severity of acne is not only dependent on the proliferation of P. acnes but also dependent on the pro-inflammatory potential of P. acnes strains and consequently constitutes potential triggering factor for acne scarring. We investigated pro-inflammatory potential of five different strains of P. acnes and P. avidum in skin explants and the preventive effect of zinc gluconate. The expression of immune markers was studied by immunohistochemistry, RT-qPCR and ELISA. P. acnes strains modulate differently the expression of immune markers both at gene and at protein levels. P. acnes type III had the highest pro-inflammatory potential by up-regulating the expression of PAR-2, TNF-alpha, MMP-13 and TIMP-2, whereas P. avidum had the weakest by up-regulating only MMP-13 and TIMP-2. Preincubation of zinc gluconate, which is a modulator of innate immunity, down-regulates the expression of most immune markers induced by P. acnes, PAR-2, TIMP-2, up-regulates MMP-1, TIMP-1. Our results demonstrate that different P. acnes strains have different inflammatory potential targeting markers of cutaneous innate immunity, and that inflammatory potential can be down-regulated by zinc gluconate. As such, the inflammatory potential of P. acnes strains on acne skin may influence the severity of inflammatory acne lesions and scars.

Photodynamic therapy with methyl-aminolevulinic acid for paucilesional mycosis fungoides: a prospective open study and review of the literature.

BACKGROUND: Publications reporting photodynamic therapy (PDT) in mycosis fungoides (MF) are rare, involve small samples, and are difficult to compare because of a lack of technical standardization. OBJECTIVE: We sought to assess PDT effectiveness and tolerability in early-stage MF using a strict reproducible procedure. METHODS: This was a prospective study conducted in Nantes University Hospital, France, including patients older than 18 years with histologically proven MF (stage IA or IB). Methyl-aminolevulinic acid-PDT sessions were repeated monthly for 6 months. Clinical and histologic responses were assessed 1 month after the last session. Patient satisfaction was assessed by telephone survey. RESULTS: Twelve patients (with 29 lesions) were treated with PDT. An objective response in target lesions was obtained in 75% of patients. Response rates were similar between plaques and patches but higher in sun-protected compared with sun-exposed areas (trend without reaching significance). During PDT, new lesions appeared in 5 of 12 patients in untreated areas. Most patients were highly satisfied and preferred PDT to the topical chemotherapy previously used. LIMITATIONS: PDT procedure criteria selection was partially arbitrary. CONCLUSIONS: In early-stage MF, PDT is effective and appreciated (especially when compared with conventional topical chemotherapy). Unilesional and paucilesional forms and lesions in sun-protected areas are to be preferred.

Zinc gluconate is an agonist of peroxisome proliferator-activated receptor-α in the epidermis.

Peroxisome proliferator-activated receptors-α (PPARs-α) are nuclear receptors with anti-inflammatory properties. Zinc gluconate is efficient in the treatment of several inflammatory dermatoses. The aim of our work was to determine whether the modulation of PPAR-α expression and activity could be one of the mechanisms of action of zinc gluconate anti-inflammatory activity in inflammatory dermatoses. Thus, we used ex vivo skin explants incubated with lipopolysaccharide (LPS), a pro-inflammatory molecule, with or without zinc gluconate. We evaluated PPAR-α protein expression using immunohistochemistry, PPAR-α DNA-binding activity using an ELISA-like technique, and PPAR-α mRNA levels using quantitative PCR. On the one hand, we found that PPAR-α epidermal protein expression was stimulated by LPS and that LPS suppressed PPAR-α mRNA expression, without modifying its function. On the other hand, in inflammatory LPS-stimulated explants, zinc gluconate significantly upregulated PPAR-α function and mRNA expression level, without changing its epidermal protein expression. These results suggest that zinc gluconate may be a PPAR-α agonist, which might play a role in the anti-inflammatory activity of this molecule.

Predictive factors of response to vismodegib: a French study of 61 patients with multiple or locally advanced basal cell carcinoma

Background: Vismodegib is indicated for the treatment of advanced or metastatic basal cell carcinoma (BCC). The predictive factors of response to vismodegib have so far been poorly described. Objectives: The primary objective was to determine the profile of patients responding to vismodegib and the duration of response. Secondary objectives were to assess whether there is a correlation between the duration of treatment and the risk of relapse, and to define factors associated with relapse. Materials & Methods: We included 61 patients with locally advanced BCC (laBCC) or multiple BCC, treated with vismodegib (150 mg per day), from July 2011 to November 2015, in the Oncodermatology Department of Nantes University Hospital in France. Tumour response was assessed using Response Evaluation Criteria in Solid Tumours version 1.1. Results: Thirty-nine patients had advanced BCC (64%) and 22 patients multiple BCC (36%), including 10 patients with Gorlin syndrome. No factor predicted response to vismodegib. The median progression-free survival (PFS) was 69.5 months for the total population. In multivariate analysis, multiple BCC was the only factor associated with an increased risk of relapse (HR: 13.80 [CI95%, 1.93-98.64, p < 0.01]). Treatment duration decreased the risk of relapse (HR 0.95 [CI95%, 0.90-0.99, p = 0.0467]). Among the 20 patients who experienced relapse during follow-up, 15 (75%) were re-treated with vismodegib, with a response rate of 66%. Conclusion: Although we were unable to establish predictive factors for the response to vismodegib, we demonstrate for the first time that increased treatment duration correlates with a decreased risk of relapse.

Ex vivo demonstration of a synergistic effect of Adapalene and benzoyl peroxide on inflammatory acne lesions.

Acne is a chronic inflammatory disease of the pilosebaceous follicle. Thanks to its ability to reduce both comedones and inflammatory lesions, the association of a retinoid and benzoyl peroxide (BPO) is now recommended for the treatment of acne. However, the mechanisms of action of this combined therapy on inflammatory acne lesions are not well understood. In an ex vivo immunohistochemistry study, we investigated the potential synergistic modulator effect of Adapalene associated with BPO on keratinocytes proliferation/differentiation and innate immunity in inflammatory acne lesions. We demonstrated that proliferation (Ki-67), adhesion/differentiation (integrin α(2), α(3) and α(6)) and innate immunity (TLR-2, ß-defensin 4, IL-8) markers are overexpressed in inflammatory acne skin compared with uninvolved acne skin. Association of Adapalene and BPO significantly decreased expression of Ki67, α(2) and α(6) integrins, TLR-2, ß-defensin 4 and IL-8 in inflammatory acne skin, whereas single treatments with Adapalene or BPO alone were less effective. These results contribute to explain the comedolytic and anti-inflammatory activities of this combined therapy observed in recent clinical trials.

Systemic rituximab in multifocal primary cutaneous follicle centre lymphoma.

The aim of this retrospective study was to assess the efficacy and tolerance of intravenous rituximab in multifocal primary cutaneous follicle centre lymphomas (PCFCL). Eleven patients with a multifocal PCFCL were treated with rituximab (MabThera(®)) administrated intravenously. After four infusions, an objective response was observed in 90% of patients, and one month after all the infusions (median of 6 infusions) all the patients had an objective response and complete remission was obtained in 7 of 11 patients (64%). Follow-up ranged from 9 to 65 months (median: 30 months). Local disease recurrence was observed in five patients. The median progression-free survival time after the end of treatment was 23.6 months. This study is the largest series of patients with multifocal primary PCFCL treated with intravenous rituximab. This therapy is a safe and effective treatment and could represent an excellent alternative treatment to radiotherapy.

TLR expression in human melanoma cells.

UNLABELLED: There is increasing evidence that melanoma cells express TLR2, -3, and -4. However, the expression of other TLRs by these cells still remains unknown. We investigated the expression patterns of TLR2, -3, -4, -7, -8 and -9 both on melanoma-invaded lymph nodes and on melanoma cell lines. TLR2, -3, -4, -7 and -9 mRNA expression was determined by quantitative RT-PCR. The TLR protein expression level was measured ex vivo by immunohistochemistry and in vitro by flow cytometry. RESULTS: At the mRNA level, melanoma cells in vitro, and possibly ex vivo, expressed TLR2, -3, -4, -7 and -9. TLR2 and -4 protein expressions ex vivo were over 50%, contrasting with an absence of these 2 TLRs in vitro. On the contrary, TLR-3 and -8 proteins had a low expression ex vivo with a high expression in vitro. TLR-7 and -9 proteins were expressed ex vivo and in vitro. Our study demonstrates for the first time that melanoma cells express TLR7 and -8.

Circulating Tumor DNA Early Kinetics Predict Response of Metastatic Melanoma to Anti-PD1 Immunotherapy: Validation Study.

The ability of early (first weeks of treatment) ctDNA kinetics to identify primary resistance to anti-PD1 immunotherapies was evaluated with a validation cohort of 49 patients treated with anti-PD1 for metastatic BRAF or NRAS-mutated melanoma, alone and pooled with the 53 patients from a previously described derivation cohort. BRAF or NRAS mutations were quantified on plasma DNA by digital PCR at baseline and after two or four weeks of treatment. ctDNA kinetics were interpreted according to pre-established biological response criteria. A biological progression (bP, i.e., a significant increase in ctDNA levels) at week two or week four was associated with a lack of benefit from anti-PD1 (4-month PFS = 0%; 1-year OS = 13%; n = 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%; 1-year OS = 73%; n = 26/102), as did patients whose ctDNA kinetics were not evaluable, due to low/undetectable baseline ctDNA (4-month PFS = 80%; 1-year OS = 81%; n = 64/102). ctDNA detection at first-line anti-PD1 initiation was an independent prognostic factor for OS and PFS in multivariate analysis. Overall, early ctDNA quantitative monitoring may allow the detection of primary resistances of metastatic melanoma to anti-PD1 immunotherapies.

About the cutaneous targets of bexarotene in CTCL patients.

There are several approved therapies for cutaneous T-cell lymphoma (CTCL). The retinoids are one of the major biologic response modifiers used in CTCL, producing good response rates but few complete responses. Bexarotene has been demonstrated to act on malignant T-cells by inducing their apoptosis, but nothing is known about its role on keratinocytes and Langerhans cells. Immunohistochemical analysis using CD1a, HLA-DR, ICAM-1 (activation markers), CD95 and CD40 (apoptosis markers) was conducted on frozen sections of bexarotene-exposed cutaneous explants and skin biopsy specimens from patients treated with bexarotene. None of the studied markers was significantly modulated both on cutaneous explants and on skin biopsy specimens after treatment with bexarotene, compared to controls. Langerhans cells and keratinocytes do not appear to play a central role in the therapeutic control of CTCL by bexarotene therapy. The main bexarotene's target thus remains T-cells by inducing their apoptosis, a mechanism that is different from the other retinoids used in CTCL.

Absence of modulation of CD4+CD25 regulatory T cells in CTCL patients treated with bexarotene.

Cutaneous T-cell lymphoma (CTCL) are a heterogeneous group of lymphoproliferative disorders, characterized by the infiltration of the epidermis by mature and activated malignant CD4+ T-lymphocytes. Retinoids such as retinoic acid and synthetic analogues have long been used alone or in combination with other therapies for CTCL. Bexarotene, the first synthetic highly selective RXR retinoid, was approved for the treatment of all stages of CTCL in patients refractory to at least one systemic therapy. Recently, six cases in which the initiation of bexarotene therapy for CTCL was associated with the progression of internal disease despite improvement of cutaneous signs and symptoms were reported. Moreover, it has been established that retinoids promote the generation of CD4+ Foxp3+ regulatory T cells, raising the question of an induction of regulatory T-cells by bexarotene. The aim of this work was to determine if bexarotene induces an increase of functional regulatory T cells which could play a role in the development of secondary extra-cutaneous lymphomas. Regulatory T cells were studied both in cutaneous biopsy specimens using an immunohistochemical analysis of CD4, CD25 and Foxp3 and in blood where proportion and functionality of circulating CD4+CD25(high) T-cells were determined. The study was performed in 10 patients [five patients with Sézary syndrome (SS) and five mycosis fungoïdes (MF)], treated for 6 months with bexarotene. Four healthy donors were used as controls for phenotypic and functional analysis on PBL. We found that the frequency of CD4+CD25(high) Treg cells was not significantly different before starting bexarotene and after 6 months of treatment in CTCL patients. However, we observed that the frequency of CD4+CD25(high) Treg cells before the beginning of the treatment was significantly increased compared to healthy donors. In addition, functional assays demonstrated that Foxp3 expressing CD4+CD25(high) T-cells were capable of suppressing autologous CD4 + CD25- T-cell proliferation. In the present work, we detected the presence of functional circulating CD4+CD25(high) Foxp3+ regulatory T-cells in CTCL patients, with an increased frequency compared to healthy donors. The treatment with bexarotene does not seem to affect the regulatory T-cell compartment.