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.

PD-L1 expression by tumor cell lines: A predictive marker in melanoma.

Prognostic biomarkers for patients with melanoma after lymph node resection are of clinical relevance and could thus enable the identification of patients who therefore would most benefit from adjuvant treatment. The aim of this work was to determine, using an in vitro model, whether immune-related biomarkers, such as MHC-class I and II, melanoma-associated antigens, IDO1 and PD-L1, could also be relevant to predict the risk of relapse of patients with stage III melanoma after lymph node resection. We established tumor cell lines from metastatic lymph nodes of 50 patients with melanoma. The expression of investigated biomarkers was determined on untreated and IFN-γ treated melanoma cell lines using flow cytometry. Among the selected biomarkers, the IFN-γ-induced expression of PD-L1 and IDO1 was associated with an increased risk of relapse (P = .0001 and P = .013, respectively) and was also associated with death for IDO1 (P = .0005). In the future, this immunologic signature could permit the identification of patients at higher risk of relapse and justifying an adjuvant treatment using immunotherapy.

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.

Value of large scale expansion of tumor infiltrating lymphocytes in a compartmentalised gas-permeable bag: interests for adoptive immunotherapy.

BACKGROUND: Adoptive cell therapy (ACT) has emerged as an effective treatment for patients with metastatic melanoma. However, there are several logistical and safety concerns associated with large-scale ex vivo expansion of tumour-specific T lymphocytes for widespread availability of ACT for cancer patients. To address these problems we developed a specific compartmentalised bag allowing efficient expansion of tumour-specific T lymphocytes in an easy handling, closed system. METHODS: Starting from lymph nodes from eight melanoma patients, we performed a side-by-side comparison of Tumour-Infiltrating Lymphocytes (TIL) produced after expansion in the compartmentalised bag versus TIL produced using the standard process in plates. Proliferation yield, viability, phenotype and IFNγ secretion were comparatively studied. RESULTS: We found no differences in proliferation yield and cell viability between both TIL production systems. Moreover, each of the cell products complied with our defined release criteria before being administered to the patient. The phenotype analysis indicated that the compartmentalised bag favours the expansion of CD8+ cells. Finally, we found that TIL stimulated in bags were enriched in reactive CD8+ T cells when co-cultured with the autologous melanoma cell line. CONCLUSIONS: The stimulation of TIL with feeder cells in the specifically designed compartmentalised bag can advantageously replace the conventional protocol using plates. In particular, the higher expansion rate of reactive CD8+ T cells could have a significant impact for ACT.

Defective activations of STAT3 Ser727 and PKC isoforms lead to oncostatin M resistance in metastatic melanoma cells.

Stage III melanoma is refractory to common therapies and shows resistance to the anti-proliferative activity of cytokines in vitro. We previously demonstrated that, for 30% of the metastatic melanoma cell lines, oncostatin M (OSM) resistance is due to the epigenetic silencing of its receptor OSMRbeta. Here we analyse, on a larger panel of short-term cultures derived from melanoma-invaded lymph nodes, other mechanisms potentially implicated in OSM resistance. For 18% of the cell lines, OSM resistance is associated with a phosphorylation defect of signal transducer and activator of transcription (STAT)3 on serine (Ser)727, in concordance with defects in the activation of various protein kinase C (PKC) isoforms, especially PKCdelta. For 21% of the cell lines, OSM resistance is associated with a defect in the activation of Akt on Ser473. By the use of inhibitors, dominant negatives and small interfering (si)RNA, we show that the PKC-STAT3 Ser727, but not the Akt, pathway appears necessary for OSM anti-proliferative activity. Moreover, we bring evidence that OSM or interleukin (IL)-6, produced in lymph nodes and/or melanoma cells, could be involved in the establishment of OSM resistance during melanoma progression. These findings could be relevant for the prognosis and the treatment of stage III melanoma patients.

Absence of amplification of CD4+CD25(high) regulatory T cells during in vitro expansion of tumor-infiltrating lymphocytes in melanoma patients.

The exact role of CD4+CD25(high) regulatory T cells (Treg) in adoptive immunotherapy of melanoma is still to be determined, and an association between an expansion of Treg cells and the expansion of therapeutic tumor-infiltrating lymphocytes (TIL) remains unelucidated. In this context, the aim of our study was to determine whether functional Treg cells were detectable and amplified among in vitro-expanded TIL from 10 metastatic melanoma lymph nodes (LNs). In this study, we investigated the expression of forkhead/winged helix transcription factor 3 (Foxp3) in melanoma-invaded LNs and determined proportion and functionality of Treg cells among TIL extracted from these 10 metastatic melanoma LNs at different steps of their in vitro expansion. We found that metastatic melanoma LNs expressed very heterogeneous levels of Foxp3 and that CD4+CD25(high) Treg cells extracted from these LNs were detectable at each step of the in vitro culture of TIL but decreasing during the culture. In addition, functional assays demonstrated that these CD4+CD25(high) T cells were capable of suppressing autologous CD8+ and CD4+CD25- T cell proliferation. These cells were indeed Treg cells as they expressed Foxp3. In conclusion, our work suggests that CD4+CD25(high) Foxp3 expressing T cells are not expanded during in vitro amplification of TIL obtained from melanoma-invaded LNs.

Adoptive Cell Therapy with Tumor-Infiltrating Lymphocytes in Advanced Melanoma Patients.

Immunotherapy for melanoma includes adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TILs). This monocenter retrospective study was undertaken to evaluate the efficacy and safety of this treatment of patients with advanced melanoma. All advanced melanoma patients treated with TILs using the same TIL expansion methodology and same treatment interleukin-2 (IL-2) regimen between 2009 and 2012 were included. After sterile intralesional excision of a cutaneous or subcutaneous metastasis, TILs were produced according to a previously described method and then infused into the patient who also received a complementary subcutaneous IL-2 regimen. Nine women and 1 man were treated for unresectable stage IIIC (n = 4) or IV (n = 6) melanoma. All but 1 patient with unresectable stage III melanoma (1st line) had received at least 2 previous treatments, including anti-CTLA-4 antibody for 4. The number of TILs infused ranged from 0.23 × 109 to 22.9 × 109. Regarding safety, no serious adverse effect was reported. Therapeutic responses included a complete remission, a partial remission, 2 stabilizations, and 6 progressions. Among these 4 patients with clinical benefit, 1 is still alive with 9 years of follow-up and 1 died from another cause after 8 years of follow-up. Notably, patients treated with high percentages of CD4 + CD25 + CD127lowFoxp3+ T cells among their TILs had significantly shorter OS. The therapeutic effect of combining TILs with new immunotherapies needs further investigation.

Tissue prognostic markers for adoptive immunotherapy in melanoma.

Adoptive immunotherapy for melanoma appears to be a promising approach. The aim of our study was to discuss the role of "tumour tissue" immunogenicity in response to adoptive immunotherapy. We thus studied the potential correlation between the expression of some melanocyte differentiation antigens, adhesion molecules and cytokines expressed by the tumour cells and the survival of patients receiving an adoptive immunotherapy with autologous Tumour Infiltrating Lymphocytes (TIL). An immunohistochemical study was performed on the lymph node samples obtained from 38 patients who received autologous TIL plus interleukin-2. Frozen sections were immunostained for melanocyte differentiation antigens (Melan-A and gp100), MHC molecules (Class I and II), adhesion molecules (ICAM-1, LFA-3) and suppressive cytokines (IL-10, TGF-beta and alpha-MSH). Expression levels of each marker were evaluated using a semi-quantitative visual scale. Using a multivariate analysis, a low expression level of TGF-beta by tumour cells was significantly associated with a prolonged relapse-free survival. A low expression level of TGF-beta, IL-10, ICAM-1 and alpha-MSH by tumour cells was significantly associated with a longer overall survival. This work suggests that a weak expression of immunosuppressive cytokines (IL-10, TGF-beta and alpha-MSH) could be a favourable prognostic marker for patients receiving autologous TIL.

CD8(+) T lymphocytes reactive against Epstein-Barr virus antigens in skin lesions of a patient with Sézary syndrome.

Several studies have investigated the possible involvement of viral agents, and among them herpes viruses, in the development of cutaneous T-cell lymphoma. The aim of our study was to determine whether T cells specific to Epstein-Barr virus (EBV) antigens were detectable among tumor-infiltrating lymphocytes infiltrating cutaneous lesions of a patient with Sézary syndrome. To analyze responses to EBV, we used a transient SV-40 origin-defective transformed simian cells transfection assay that permits an estimation of CD8 T-cell responses against a large number of HLA/viral protein combinations. This technique allowed the detection of EBV-specific T lymphocytes mainly directed against epitopes generated during the lytic cycle in the cutaneous lesions. This is, to our knowledge, the first description of the presence of EBV-specific T lymphocytes among tumor-infiltrating lymphocytes infiltrating the lesional skin of a patient with Sézary syndrome.

[Passive immunotherapy of melanoma]. / L'immunothérapie passive du mélanome.

The protocols of tumor immunotherapy have been largely developed in the past ten years due to the identification of many tumor antigens recognized specifically by CD8 and CD4 T lymphocytes. Among the various immunotherapies currently tested, passive immunotherapy (i.e injection of T lymphocytes generated and selected ex-vivo from blood or from the tumor), seems to be particularly promising. Indeed, three recent studies evidence the efficacy of such therapy in melanoma treatment. For the first time, a precise immunological follow-up was carried out, thus showing the correlation between the therapeutic benefit and the injection of tumor antigen specific T lymphocytes, and the survival and the preferential migration of these lymphocytes to the tumor sites. Although all the questions about optimal methods for this mode of therapy are not solved, these recent results constitute a major breakthrough in the field of the tumor immunotherapy.

Adoptive TIL transfer in the adjuvant setting for melanoma: long-term patient survival.

Two first analyses of our clinical trial on TIL as adjuvant therapy for melanoma were published in 2002 and 2007. We present here an update of the clinical results after a 17-year median followup. In this trial, disease-free patients were randomly assigned to receive either TIL/IL-2 or IL-2. The relapse-free survival (RFS) was the primary objective. Eighty-eight patients were enrolled. A new analysis performed in May 2013 did not show significant changes in RFS or OS duration. However, our first finding on the association between the number of invaded lymph nodes and TIL effectiveness was strengthened. The Cox model adjusted on this interaction showed for the first time a significant treatment effect when considering the overall population, both on the RFS and OS. Patients treated with TIL had a longer RFS (P = 0.023) or OS (P = 0.020). This study being with a very long followup (17 years), confirmed the association between TIL effectiveness and the number of invaded lymph nodes, indicating that a low tumor burden could be a crucial factor enhancing the curative effect of TIL in possible microscopic residual disease. Moreover, we confirmed that a prolonged survival was associated with the presence of specific TIL and a decrease in Foxp3 expression.

Tissue biomarkers in melanoma patients treated with TIL.

While treating stage III melanoma patients with autologous therapeutic TIL in an adjuvant setting, we previously reported a significant benefit of treatment on both progression-free survival and overall survival in patients with only one invaded lymph node (early stage III) compared to patients with more than one invaded lymph nodes (advanced stage III). In this context, in order to understand the difference of activity of TIL therapy according to the progression of the illness at stage III, the first objective of the present study was to determine potential differences in the characteristics of TIL populations obtained from an early stage III and a more advanced stage III when tumor burden is more important. The second objective was to determine possible differences in tissue expression level of several molecules involved in interactions between tumor cells and T cells between early and advanced stage III considering that the tumor microenvironment of invaded lymph nodes could become more tolerant with the progression of the disease. A total of 47 samples of melanoma invaded LN from stage IIIb (AJCC 2007) melanoma patients treated with TIL plus IL-2 were included in this study. We confirmed that both PFS and OS were significantly associated to the presence of tumor-reactive T-cells among TIL injected to the patients and that these tumor reactive T cells were more frequently observed at the early stage III. Moreover, while analyzing the expression of 17 markers on 34/47 tumor specimens using immunohistochemistry, we identified that 3 tissue markers involved in interactions between melanoma cells and T cells have a significant difference of expression between early and advanced stage III: MHC class I, adhesion molecule ICAM-1 and the co-stimulation molecule LFA-3 had a significantly weaker expression in melanoma tissue specimens from advanced stage III. In addition, the expression of the alpha chain of the IL-2 receptor (CD25) and the nuclear transcription factor Foxp3 was significantly increased in the melanoma tissue specimens from advanced stage III. Our results suggest differences in the immunological status of the tumor microenvironment between early and advanced stage III, which could explain the difference in clinical response to TIL infusion in an adjuvant setting between early and advanced stage III.

Treatment of metastatic melanoma with autologous Melan-A/MART-1-specific cytotoxic T lymphocyte clones.

Immunotherapy by adoptive T-cell transfer aims at maximizing tumor antigen-specific T-cell responses. We treated 14 patients at the metastatic stage in a phase II study with Melan-A-specific T-cell clones generated from patient blood. During the period required for T-cell clone generation, the patients were treated by dacarbazine. Every patient received a T-cell clone suspension followed by subcutaneous injections of interleukin 2 and interferon alpha. Patients were monitored until disease progression occurred. We succeeded in obtaining autologous Melan-A-specific cytotoxic T lymphocyte clones, which were highly reactive against tumor cells for all the patients. Of the 14 patients treated, six (43%) experienced an objective response (CR + PR) with long-term complete remission for two patients (1 CR for 5 years and 1 CR for 28 months). Furthermore, we showed that all the clinical responses were significantly associated with in vivo expansion of the Melan-A-specific T-cell repertoire. This phenomenon appeared to be significantly associated with clinical responses. Thus, over the course of an adoptive cell transfer, monitoring this melanoma-specific T-cell expansion in patient blood appears crucial for predicting the clinical efficiency of such an immunological approach.

Infusion of Melan-A/Mart-1 specific tumor-infiltrating lymphocytes enhanced relapse-free survival of melanoma patients.

Adoptive therapy of cancer has been mostly tested in advanced cancer patients using tumor-infiltrating lymphocytes (TIL). Following discouraging results likely due to poor tumor-specificity of TIL and/or high tumor burden, recent studies reiterate the enormous potential of this therapy, particularly in melanoma. We had performed a phase II/III randomised trial on 88 stage III melanoma patients, who received autologous TIL plus IL-2 or IL-2 alone, after complete tumour resection. We reported previously clinical and immunological results supporting the ability of tumor reactive TIL infusion to prevent further development of the melanoma disease and to increase overall survival of patients bearing only one tumor invaded lymph node. The absence of correlation between overall and disease-free survival and the amount of infused tumor-specific TIL suggested that therapeutic efficiency might depend on other parameters such as antigen specificity, function or persistence of TIL. Here we studied the recognition of a panel of 38 shared tumor-associated antigens (TAA) by TIL infused to the patients included in this assay, in order to determine if treatment outcome could correlate with particular antigen specificities of infused TIL. Results show that the infusion of Melan-A/MART-1 reactive TIL appears to be associated with a longer relapse-free survival for HLA-A2 patients. These results further support the relevance of Melan-A/MART-1 antigen as a prime target for immunotherapy protocols in melanoma.

Long-term follow-up of patients treated by adoptive transfer of melanoma tumor-infiltrating lymphocytes as adjuvant therapy for stage III melanoma.

The first analysis of our clinical trial on interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma was published in 2002 [5]. The aim of this paper is to update clinical results of 7 years of follow-up after the last treated patient. In the trial conducted between December 1993 and January 1999, patients without any detectable metastases after lymph node excision were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The duration of the relapse-free interval was the primary objective. Eighty-eight patients were enrolled in the study. Currently, the last analysis performed in June 2006, after a median follow-up of 114.8 months, did not show change of non-significant extension of the relapse-free interval or overall survival. However, this second analysis strengthens our first hypothesis about the relationship between number of invaded lymph nodes and TIL treatment effectiveness. In the group with only one invaded lymph node, the estimated relapse rate was significantly lower (P (adjusted) = 0.0219) and the overall survival was increased (P (adjusted) = 0.0125) in the TIL+IL-2 arm compared with the IL-2 only arm. No differences between the two arms, either with regard to the duration of disease-free survival (P (adjusted) = 0.38) or overall survival (P (adjusted) = 0.43), were noted in the group with more than one invaded lymph node, whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study, with a very long follow up (median of almost 10 years), postulates for the first time relationship between TIL efficiency in stage III melanoma (AJCC) and number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the production of an effective in vitro expansion of T cells specific for autologous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma.

Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells.

In this study, we report the adoptive transfer of highly tumor-reactive Melan-A-specific T cell clones to patients with metastatic melanoma, and the follow-up of these injected cells. These clones were generated from HLA-A*0201 patients by in vitro stimulations of total PBMC with the HLA-A*0201-binding Melan-A peptide analog ELAGIGILTV. Ten stage IV melanoma patients were treated by infusion of these CTL clones with IL-2 and IFN-alpha. The generated T cell clones, of effector/memory phenotype were selected on the basis of their ability to produce IL-2 in response to HLA-A*0201 Melan-A-positive melanoma lines. Infused clones were detected, by quantitative PCR, in the blood of three patients for periods ranging from 7 to 60 days. Six patients showed regression of individual metastases or disease stabilization, and one patient experienced a complete response, but no correlation was found between the detection of the infused clones in PBMC or tumor samples and clinical responses. Nonetheless, frequencies of Melan-A/A2-specific lymphocytes, measured by tetramer labeling, increased after treatment in most patients. In one of these patients, who showed a complete response, this increase corresponded to the expansion of new clonotypes of higher avidity than those detected before treatment. Together, our results suggest that infused CTL clones may have initiated an antitumor response that may have resulted in the expansion of a Melan-A-specific CTL repertoire.

Expression of CD94/NKG2-A on human T lymphocytes is induced by IL-12: implications for adoptive immunotherapy.

NK cell receptors (NKRs) are expressed on a subset of human T cells, predominantly CD8(+), within which they can modulate TCR-mediated functions. In an attempt to identify the mechanisms leading to NKR expression, we analyzed the capacity of IL-12 to modulate the expression by T cells of the components of the CD94/NKG2-A inhibitory receptor, a member of the C-type lectin-like family of NKR. We show that IL-12 induces the expression of NKG2-A and/or CD94 by CD8(+) T cells in culture, and that this induction was mediated neither by IFN-gamma nor by IL-15. We also show, using the redirected killing assay, that IL-12-induced expression of both CD94 and NKG2-A led to the acquisition by T cells of a functional inhibitory receptor. Expression of the CD94/NKG2-A inhibitory receptor was also induced by IL-12 during T cell Ag stimulation so that in the presence of this cytokine a high proportion of melanoma-reactive CTL induced from PBL by melanoma peptide stimulation expressed this receptor. This study emphasizes the implication of IL-12 in the modulation of immune responses through NKR induction.