Melanoma: Liénard D

Guggisberg D, Cerottini JP, Krischer J, Braun R, Dietrich PY, Liénard D, Anonymous00229. · Département hospitalo-universitaire romand de dermatologie et vénéréologie, Lausanne et Genève. · Rev Med Suisse Romande. · Pubmed #11887568 No free full text.

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Lejeune FJ, Liénard D, Matter M, Rüegg C. · Multidisciplinary Oncology Centre (CePO), Lausanne, Switzerland. · Cancer Immun. · Pubmed #16551058 links to  free full text

Abstract: Recombinant human tumour necrosis factor (TNF) has a selective effect on angiogenic vessels in tumours. Given that it induces vasoplegia, its clinical use has been limited to administration through isolated limb perfusion (ILP) for regionally advanced melanomas and soft tissue sarcomas of the limbs. When combined with the alkylating agent melphalan, a single ILP produces a very high objective response rate. In melanoma, the complete response (CR) rate is around 80% and the overall objective response rate greater than 90%. In soft tissue sarcomas that are inextirpable, ILP is a neoadjuvant treatment resulting in limb salvage in 80% of the cases. The CR rate averages 20% and the objective response rate is around 80%. The mode of action of TNF-based ILP involves two distinct and successive effects on the tumour-associated vasculature: first, an increase in endothelium permeability leading to improved chemotherapy penetration within the tumour tissue, and second, a selective killing of angiogenic endothelial cells resulting in tumour vessel destruction. The mechanism whereby these events occur involves rapid (of the order of minutes) perturbation of cell-cell adhesive junctions and inhibition of alphavbeta3 integrin signalling in tumour-associated vessels, followed by massive death of endothelial cells and tumour vascular collapse 24 hours later. New, promising approaches for the systemic use of TNF in cancer therapy include TNF targeting by means of single chain antibodies or endothelial cell ligands, or combined administration with drugs perturbing integrin-dependent signalling and sensitizing angiogenic endothelial cells to TNF-induced death.

Speiser DE, Pittet MJ, Rimoldi D, Guillaume P, Luescher IF, Liénard D, Lejeune F, Cerottini JC, Romero P. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, Centre Hospitalier Universitaire Vaudois, Niveau 5, Aile Est, Av. Pierre-Decker 4, CH-1005 Lausanne, Switzerland. · Semin Cancer Biol. · Pubmed #15001165 No free full text.

Abstract: Immunotherapy of melanoma is aimed to mobilize cytolytic CD8+ T cells playing a central role in protective immunity. Despite numerous clinical vaccine trials, only few patients exhibited strong antigen-specific T-cell activation, stressing the need to improve vaccine strategies. For a rational development, we propose to focus on molecularly defined vaccine components, and evaluate their immunogenicity with highly reproducible and standardized methods for ex vivo immune monitoring. Careful immunogenicity comparison of vaccine formulations in phase I/II studies allow to select optimized vaccines for subsequent clinical efficacy testing in large scale phase III trials.

Romero P, Valmori D, Pittet MJ, Zippelius A, Rimoldi D, Lévy F, Dutoit V, Ayyoub M, Rubio-Godoy V, Michielin O, Guillaume P, Batard P, Luescher IF, Lejeune F, Liénard D, Rufer N, Dietrich PY, Speiser DE, Cerottini JC. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne branch, University Hospital (CHUV), Lausanne, Switzerland. · Immunol Rev. · Pubmed #12445283 No free full text.

Abstract: Some cancer patients mount spontaneous T- and B-cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T-cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure-function properties of the melanocyte/melanoma tumor antigen Melan-A/MART-1, the assessment of the T-cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine-induced CTL responses to this antigen in patients with metastatic melanoma.

Lejeune FJ, Kroon BB, Di Filippo F, Hoekstra HJ, Santinami M, Liénard D, Eggermont AM. · Multidisciplinary Oncology Center, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. · Surg Oncol Clin N Am. · Pubmed #11641093 No free full text.

Abstract: Isolated limb perfusion (ILP) is a method of cancer treatment allowing the administration of high doses of anticancer agents in a limb surgically isolated from systemic circulation. By using continuous leakage monitoring and using the drug melphalan, a high complete remission rate is obtained in patients with melanoma. In patients with sarcomas, ILP with tumor necrosis factor and melphalan represents a neoadjuvant treatment for limb-sparing surgery. This treatment is the first demonstration of an active anti-angiogenic regimen in the clinic.

van Baren N, Bonnet MC, Dréno B, Khammari A, Dorval T, Piperno-Neumann S, Liénard D, Speiser D, Marchand M, Brichard VG, Escudier B, Négrier S, Dietrich PY, Maraninchi D, Osanto S, Meyer RG, Ritter G, Moingeon P, Tartaglia J, van der Bruggen P, Coulie PG, Boon T. · Ludwig Institute for Cancer Research, 74 avenue Hippocrate, UCL7459, B-1200 Brussels, Belgium; e-mail: · J Clin Oncol. · Pubmed #16061912 No free full text.

Abstract: PURPOSE: To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3(168-176) and MAGE-1(161-169), which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). MATERIALS AND METHODS: The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3(168-176) and MAGE-1(161-169) peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. RESULTS: Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. CONCLUSION: Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient.

Speiser DE, Liénard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F, Krieg AM, Cerottini JC, Romero P. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Multidisciplinary Oncology Center, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. · J Clin Invest. · Pubmed #15696196 links to  free full text

Abstract: The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.

Liénard D, Rimoldi D, Marchand M, Dietrich PY, van Baren N, Geldhof C, Batard P, Guillaume P, Ayyoub M, Pittet MJ, Zippelius A, Fleischhauer K, Lejeune F, Cerottini JC, Romero P, Speiser DE. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. · Cancer Immun. · Pubmed #15149168 links to  free full text

Abstract: The purpose of this study was to test melanoma vaccines consisting of peptides and immunological adjuvants for optimal immunogenicity and to evaluate laboratory immune monitoring for in vivo relevance. Forty-nine HLA-A2 positive patients with Melan-A positive melanoma were repeatedly vaccinated with Melan-A peptide, with or without immune adjuvant AS02B (QS21 and MPL) or IFA. Peptide-specific CD8 T cells in PBLs were analyzed ex vivo using fluorescent HLA-A2/Melan-A multimers and IFN-gamma ELISPOT assays. The vaccines were well tolerated. In vivo expansion of Melan-A-specific CD8 T cells was observed in 13 patients (1/12 after vaccination with peptide in AS02B and 12/17 after vaccination with peptide in IFA). The T cells produced IFN-gamma and downregulated CD45RA and CD28. T-cell responses correlated with inflammatory skin reactions at vaccine injection sites (P < 0.001) and with DTH reaction to Melan-A peptide (P < 0.01). Twenty-six of 32 evaluable patients showed progressive disease, whereas 4 patients had stable disease. The two patients with the strongest Melan-A-specific T-cell responses experienced regression of metastases in skin, lymph nodes, and lung. We conclude that repeated vaccination with Melan-A peptide in IFA frequently leads to sustained responses of specific CD8 T cells that are detectable ex vivo and correlate with inflammatory skin reactions.

Kleeberg UR, Suciu S, Bröcker EB, Ruiter DJ, Chartier C, Liénard D, Marsden J, Schadendorf D, Eggermont AM, Anonymous00293. · Haematologisch-Onkologische Praxis Altona (HOPA), Max-Brauer-Allee 52, D-22765 Hamburg, Germany. · Eur J Cancer. · Pubmed #14746858 No free full text.

Abstract: Between 1988 and 1996, the European Organisation for Research and Treatment of Cancer Melanoma Group (EORTC-MG) performed a prospective, randomised phase III adjuvant trial to evaluate the efficacy and toxicity of low dose recombinant interferon-alpha 2 b (rIFN-alpha2b) (1 MU) or recombinant interferon gamma (rIFN-gamma), (0.2 mg) both given subcutaneously (s.c.), every other day (qod), for 12 months in comparison with an untreated control group. The German Cancer Society (DKG) added a fourth arm with Iscador M, a popular mistletoe extract. High-risk stage II patients (thickness >3 mm) and stage III patients (positive lymph nodes) without distant metastasis were randomised and followed until their first progression or death. An intention-to-treat analysis was performed. From 1988 to 1996, a total of 830 patients were randomised: 423 in the three-arm EORTC 18871 trial and 407 patients in the four-arm DKG 80-1 trial. The median follow-up was 8.2 years and a total of 537 relapses and 475 deaths were reported. At 8 years, the disease-free interval (DFI) rate was 32.4% and the overall survival (OS) rate was 40.0%. In terms of the DFI, the hazard ratio estimates (95% Confidence Intervals (CI)) were: 1.04 (0.84, 1.30) for the comparison of rIFN-alpha2b versus control, 0.96 (0.77, 1.20) for rIFN-gamma versus control, and 1.32 (0.93, 1.87) for Iscador M versus control. In terms of OS, the corresponding estimates (95% CI) for the 3 treatment comparisons were: for IFN-alpha2b 0.96 (0.76, 1.21), for rIFN-gamma 0.87 (0.69, 1.10) and for Iscador M 1.21 (0.84, 1.75), respectively. The results show no clinical benefit for adjuvant treatment with low dose rIFN-alpha2b or rIFN-gamma or with Iscador M in high-risk melanoma patients.

Keilholz U, Martus P, Punt CJ, Kruit W, Mooser G, Schadendorf D, Liénard D, Dummer R, Koller J, Voit C, Eggermont AM. · Medizinische Klinik III, UKBF, Free University Berlin, Hindenburgdamm 30, 12200, Berlin, Germany. · Eur J Cancer. · Pubmed #12110497 No free full text.

Abstract: The aim of this study was to define prognostic factors for survival, and especially for long-term survival in a mature data-set of patients with stage IV melanoma treated within a randomised trial of cytokine-based protocols. Long-term follow-up data on patients enrolled into a European Organization for Research and Treatment of Cancer (EORTC) trial comparing interferon-alpha (IFNalpha) plus interleukin-2 (IL-2) with or without cisplatin were collected. Univariate and multivariate Cox regression analyses were performed to define prognostic factors for survival. The characteristics of patients alive at 2 and 5 years after randomisation were compared with the entire cohort using the chi(2) test. The minimum potential follow-up of the 131 evaluable patients was 5 years. 18 patients (14%) were alive 2 years after randomisation, and 11 (8%) 5 years after randomisation. Pretreatment performance status (PS), serum lactate dehydrogenase (LDH) and tumour mass were significant predictors for survival, whereas site of metastases and number of sites were non-significant. PS and LDH were the only independent prognostic factors. All except 1 patient alive at 2 and 5 years had a pretreatment PS of 100%, and only three long-term survivors had elevated pretreatment LDH. There was no association between the site of metastases and long-term survival. Response to treatment was a major predictor for long-term survival, whereas addition of cisplatin did not impact upon overall survival probability or on long-term survival. The probability of long-term survival in stage IV melanoma patients after IL-2-based treatments is governed by pretreatment PS, serum LDH and response to treatment. Site of metastases, the basis for the M-subcategories of the new AJCC staging system, was not informative in this study.

Liénard D, Eggermont AM, Koops HS, Kroon B, Towse G, Hiemstra S, Schmitz P, Clarke J, Steinmann G, Rosenkaimer F, Lejeune FJ. · Centre Pluridisciplinaire d'Oncologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. · Melanoma Res. · Pubmed #10596916 No free full text.

Abstract: This open, multicentre, randomized phase II trial was conducted to determine the effect of isolated limb perfusion (ILP) with tumour necrosis factor-alpha (TNFalpha) in combination with melphalan with or without interferon-gamma (IFNgamma) in patients with in-transit metastases of melanoma of the limbs (MD Anderson stage IIIA or IIIAB, AJCC stage III). The 64 patients included were randomized to receive either a two- drug regimen consisting of TNFalpha and melphalan (TM-ILP) or a three-drug regimen consisting of TNFalpha, melphalan and INFgamma (TIM-ILP). Patients randomized to receive IFNgamma were pretreated for 2 days before the ILP with once daily 0.2 mg IFNgamma subcutaneously and also received the same amount of IFNgamma during ILP. A total of 47 complete responses (73%) were reported, 22 (69%) of which occurred in the TM-ILP group and 25 (78%) in the TIM-ILP group; the difference was not significant. The 14 partial responses (22%) were split evenly between the treatment groups. In the TM-ILP group, two cases of stable disease and one case of progressive disease were reported. The overall response rate (complete plus partial responses) was 100% in the TIM-ILP group and 91% in the TM-ILP group, yielding an overall response of 95% for this study. In the historical control data, where 103 patients had received melphalan alone (M-ILP), there were 54 records of complete responses (52%) and 80 of complete or partial responses (78%). The median survival time estimated by the Kaplan-Meier method was 819 days for the TM-ILP group, > 705 days for the TIM-ILP group and 873 days for the combined study population; estimates for time to local progression or recurrence were 327 days, in excess of 498 days and 405 days, respectively. The corresponding figure for the historical controls was 338 days. These data suggest that TNFalpha associated with melphalan may be superior to melphalan alone for ILP.

Gander M, Leyvraz S, Decosterd L, Bonfanti M, Marzolini C, Shen F, Liénard D, Perey L, Colella G, Biollaz J, Lejeune F, Yarosh D, Belanich M, D'Incalci M. · Centre Pluridisciplinaire d'Oncologie, Lausanne, Switzerland. · Ann Oncol. · Pubmed #10470431 links to  free full text

Abstract: BACKGROUND: The DNA repair protein O6-alkylguanine-DNA alkyl transferase (AT) mediates resistance to chloroethylnitrosoureas. Agents depleting AT such as DTIC and its new analogue temozolomide (TMZ) can reverse resistance to chloroethylnitrosoureas. We report the results of a dose finding study of TMZ in association with fotemustine. PATIENTS AND METHODS: Twenty-four patients with metastatic melanoma or recurrent glioma were treated with escalating dose of oral or intravenous TMZ ranging from 300 to 700 mg/m2, divided over two days. Fotemustine 100 mg/m2 was given intravenously on day 2, 4 hours after TMZ. AT depletion was measured in peripheral blood mononuclear cells (PBMCs) and in selected cases in melanoma metastases and was compared to TMZ pharmacokinetics. RESULTS: The maximum tolerated dose (MTD) of TMZ was 400 mg/m2 (200 mg/m2/d) when associated with fotemustine the 2nd day with myelosuppression as dose limiting toxicity. The decrease of AT level in PBMCs was progressive and reached 34% of pretreatment values on day 2. There was however wide interindividual variability. AT reduction was neither dose nor route dependent and did not appear to be related to TMZ systemic exposure (AUC). In the same patients, AT depletion in tumour did not correlate with the decrease of AT observed in PBMCs. CONCLUSIONS: PBMCs may not be used as a surrogate of tumour for AT depletion. Further study should concentrate on the pharmacokinetic pharmacodynamic relationship in tumour to provide the basis for individually tailored therapy.

Autier P, Doré JF, Négrier S, Liénard D, Panizzon R, Lejeune FJ, Guggisberg D, Eggermont AM. · Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy. · J Natl Cancer Inst. · Pubmed #10433619 links to  free full text

Abstract: BACKGROUND: In epidemiologic studies, sunscreen use is associated with increased risk of cutaneous melanoma, basal cell skin cancer, and higher numbers of nevi. It has been proposed that sunscreens may encourage prolonged sun exposure because they delay sunburn occurrence. We examined whether, under habitual conditions of sunscreen use, the sun-protection factor (SPF) had an influence on sun-exposure duration. METHODS: Before the 1997 summer holidays, we randomly assigned 87 French and Swiss participants who were 18-24 years of age to receive an SPF 10 or an SPF 30 sunscreen. Neither medical personnel nor study participants were aware of their sunscreen assignment. Participants were asked to complete daily records of their sun exposure. To avoid influencing the recreational sun-exposure habits of the study participants, no recommendation was made about sun exposure or sun protection. Furthermore, participants were told that the trial end point was the number of pigmented skin lesions before and after the holidays. One subject was lost to follow-up. All statistical tests were two-sided. RESULTS: The SPF 10 (n = 44) and SPF 30 (n = 42) groups had equivalent mean holiday durations (19.4 days versus 20.2 days) and mean quantities of sunscreen used (72.3 g versus 71.6 g). The mean cumulative sun exposures for the two groups were 58.2 hours and 72.6 hours, respectively (P =.011). The mean daily durations of sunbathing were 2.6 and 3.1 hours, respectively (P =.0013), and, for outdoor activities, they were 3.6 and 3.8 hours, respectively (P =.62). There was no difference in sunburn experience between the two groups. CONCLUSIONS: Use of higher SPF sunscreen seems to increase the duration of recreational sun exposure of young white Europeans.

Olieman AF, Liénard D, Eggermont AM, Kroon BB, Lejeune FJ, Hoekstra HJ, Koops HS. · Department of Surgical Oncology, University Hospital Groningen, The Netherlands. · Arch Surg. · Pubmed #10088573 links to  free full text

Abstract: BACKGROUND: Hyperthermic isolated limb perfusion (HILP) with tumor necrosis factor alpha (TNF-alpha), interferon gamma, and melphalan has proved to be useful in the treatment of recurrent malignant melanoma and of locally advanced soft tissue sarcomas of the extremities. OBJECTIVE: To determine whether this modality is also effective in the treatment of locally advanced nonmelanoma skin tumors of the extremities. PATIENTS AND METHODS: Fifteen patients with locally advanced primary, recurrent, or metastatic skin tumors of the extremities (12 with squamous cell carcinoma and 3 with Merkel cell carcinoma) underwent HILP with TNF-alpha, interferon gamma, and melphalan. Six tumors were localized in the upper extremity (40%), and 9 in the lower extremity (60%). Treatment-related complications, limb salvage rate, local recurrence, and regional and distant metastases were scored during a median follow-up of 20 months. RESULTS: After HILP, 9 patients (60%) showed a complete response (with histopathological confirmation). Four patients (27%) showed a partial response (with histopathological confirmation in 1 patient), and 2 patients (13%) showed no change (with histopathological confirmation in 1 patient and with clinical evidence in 1 patient). Two patients (13%) showed treatment-related complications. The limb salvage was achieved in 12 patients (80%), and the local recurrences developed in 4 patients (27%). During follow-up, regional lymph node metastases were observed in 2 patients (13%) and distant metastases in 2 patients (13%). CONCLUSION: Based on our results, HILP with TNF-alpha, interferon gamma, and melphalan should be considered as a limb-saving treatment modality in patients with locally advanced nonmelanoma skin tumors of the extremities who would otherwise be candidates for ablative surgery.

Marchand M, van Baren N, Weynants P, Brichard V, Dréno B, Tessier MH, Rankin E, Parmiani G, Arienti F, Humblet Y, Bourlond A, Vanwijck R, Liénard D, Beauduin M, Dietrich PY, Russo V, Kerger J, Masucci G, Jäger E, De Greve J, Atzpodien J, Brasseur F, Coulie PG, van der Bruggen P, Boon T. · Ludwig Institute for Cancer Research, Brussels Branch, and Université Catholique de Louvain, Belgium. · Int J Cancer. · Pubmed #9935203 No free full text.

Abstract: Thirty-nine tumor-bearing patients with metastatic melanoma were treated with 3 subcutaneous injections of the MAGE-3.A1 peptide at monthly intervals. No significant toxicity was observed. Of the 25 patients who received the complete treatment, 7 displayed significant tumor regressions. All but one of these regressions involved cutaneous metastases. Three regressions were complete and 2 of these led to a disease-free state, which persisted for more than 2 years after the beginning of treatment. No evidence for a cytolytic T lymphocyte (CTL) response was found in the blood of the 4 patients who were analyzed, including 2 who displayed complete tumor regression. Our results suggest that injection of the MAGE-3.A1 peptide induced tumor regression in a significant number of the patients, even though no massive CTL response was produced.

Willi JP, Matter M, Buchegger F, Antonescu C, Guggisberg D, Cerottini JP, Krischer J, Braun R, Marie Kurt A, Roche B, Lemoine R, Rimoldi D, Lejeune FJ, Liénard D, Bischof Delaloye A. · Service de Médecine Nucléaire, University Hospital Geneva, Rue Mucheli-du-Crest 24, 1211 Genève 14, Switzerland. · Nuklearmedizin. · Pubmed #18084679 No free full text.

Abstract: AIM: The clinical relevance of sentinel lymph node (SLN) analysis was evaluated prospectively and compared with other known risk factors of relapse in early stage melanoma. METHODS: Surgery was guided by lymphoscintigraphy, blue dye and gamma probe detection. SLN were analysed by haematoxylin eosin (HE) histochemistry and multimarker immunohistochemistry (IHC). Disease free survival (DFS) was evaluated with Kaplan-Meier plots according to different parameters and Cox analyses of variance. RESULTS: From 210 patients a total of 381 SLN were excised. Lymphoscintigraphy identified all excised SLN with only 2 false positive lymphatic lakes. Fifty patients (24%) had tumour positive SLN. With a mean follow-up of 31.3 months, 29 tumour recurrences were observed, 19 (38%) in 50 SLN positive and 10 (6%) in 160 SLN negative patients. Strong predictive factors for early relapse (p < 0.0005) were SLN positivity and a high Breslow index. CONCLUSION: SLN tumour positivity is an independent factor of high risk for early relapse with a higher power of discrimination than the Breslow index.

Matter M, Nicod Lalonde M, Allaoua M, Boubaker A, Liénard D, Gugerli O, Cerottini JP, Bouzourene H, Bischof Delaloye A, Lejeune F. · Department of Visceral Surgery, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. · J Nucl Med. · Pubmed #17873127 links to  free full text

Abstract: In sentinel node (SN) biopsy, an interval SN is defined as a lymph node or group of lymph nodes located between the primary melanoma and an anatomically well-defined lymph node group directly draining the skin. As shown in previous reports, these interval SNs seem to be at the same metastatic risk as are SNs in the usual, classic areas. This study aimed to review the incidence, lymphatic anatomy, and metastatic risk of interval SNs. METHODS: SN biopsy was performed at a tertiary center by a single surgical team on a cohort of 402 consecutive patients with primary melanoma. The triple technique of localization was used-that is, lymphoscintigraphy, blue dye, and gamma-probe. Otolaryngologic melanoma and mucosal melanoma were excluded from this analysis. SNs were examined by serial sectioning and immunohistochemistry. All patients with metastatic SNs were recommended to undergo a radical selective lymph node dissection. RESULTS: The primary locations of the melanomas included the trunk (188), an upper limb (67), or a lower limb (147). Overall, 97 (24.1%) of the 402 SNs were metastatic. Interval SNs were observed in 18 patients, in all but 2 of whom classic SNs were also found. The location of the primary was truncal in 11 (61%) of the 18, upper limb in 5, and lower limb in 2. One patient with a dorsal melanoma had drainage exclusively in a cervicoscapular area that was shown on removal to contain not lymph node tissue but only a blue lymph channel without tumor cells. Apart from the interval SN, 13 patients had 1 classic SN area and 3 patients 2 classic SN areas. Of the 18 patients, 2 had at least 1 metastatic interval SN and 2 had a classic SN that was metastatic; overall, 4 (22.2%) of 18 patients were node-positive. CONCLUSION: We found that 2 of 18 interval SNs were metastatic: This study showed that preoperative lymphoscintigraphy must review all known lymphatic areas in order to exclude an interval SN.

Roulin D, Matter M, Bady P, Liénard D, Gugerli O, Boubaker A, Bron L, Lejeune FJ. · Department of Visceral Surgery, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 46, 1011 Lausanne, Switzerland. · Eur J Surg Oncol. · Pubmed #17825518 No free full text.

Abstract: AIM: To confirm the accuracy of sentinel node biopsy (SNB) procedure and its morbidity, and to investigate predictive factors for SN status and prognostic factors for disease-free survival (DFS) and disease-specific survival (DSS). MATERIALS AND METHODS: Between October 1997 and December 2004, 327 consecutive patients in one centre with clinically node-negative primary skin melanoma underwent an SNB by the triple technique, i.e. lymphoscintigraphy, blue-dye and gamma-probe. Multivariate logistic regression analyses as well as the Kaplan-Meier were performed. RESULTS: Twenty-three percent of the patients had at least one metastatic SN, which was significantly associated with Breslow thickness (p<0.001). The success rate of SNB was 99.1% and its morbidity was 7.6%. With a median follow-up of 33 months, the 5-year DFS/DSS were 43%/49% for patients with positive SN and 83.5%/87.4% for patients with negative SN, respectively. The false-negative rate of SNB was 8.6% and sensitivity 91.4%. On multivariate analysis, DFS was significantly worsened by Breslow thickness (RR=5.6, p<0.001), positive SN (RR=5.0, p<0.001) and male sex (RR=2.9, p=0.001). The presence of a metastatic SN (RR=8.4, p<0.001), male sex (RR=6.1, p<0.001), Breslow thickness (RR=3.2, p=0.013) and ulceration (RR=2.6, p=0.015) were significantly associated with a poorer DSS. CONCLUSION: SNB is a reliable procedure with high sensitivity (91.4%) and low morbidity. Breslow thickness was the only statistically significant parameter predictive of SN status. DFS was worsened in decreasing order by Breslow thickness, metastatic SN and male gender. Similarly DSS was significantly worsened by a metastatic SN, male gender, Breslow thickness and ulceration. These data reinforce the SN status as a powerful staging procedure.

Barbey C, Baumgaertner P, Devevre E, Rubio-Godoy V, Derre L, Bricard G, Guillaume P, Luescher IF, Liénard D, Cerottini JC, Romero P, Rufer N, Speiser DE. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Centre Hospitalier Universitaire Vaudois, Avenue Pierre-Decker 4, CH-1005 Lausanne, Switzerland. · J Immunol. · Pubmed #17339453 links to  free full text

Abstract: Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

Speiser DE, Baumgaertner P, Barbey C, Rubio-Godoy V, Moulin A, Corthesy P, Devevre E, Dietrich PY, Rimoldi D, Liénard D, Cerottini JC, Romero P, Rufer N. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital of Lausanne, Lausanne, Switzerland. · J Immunol. · Pubmed #16818795 links to  free full text

Abstract: Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

Baumgaertner P, Rufer N, Devevre E, Derre L, Rimoldi D, Geldhof C, Voelter V, Liénard D, Romero P, Speiser DE. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland. · Cancer Res. · Pubmed #16488988 links to  free full text

Abstract: Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freund's adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freund's adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.

Zippelius A, Batard P, Rubio-Godoy V, Bioley G, Liénard D, Lejeune F, Rimoldi D, Guillaume P, Meidenbauer N, Mackensen A, Rufer N, Lubenow N, Speiser D, Cerottini JC, Romero P, Pittet MJ. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research and Multidisciplinary Oncology Center, University Hospital (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland. · Cancer Res. · Pubmed #15087405 links to  free full text

Abstract: Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Movassagh M, Spatz A, Davoust J, Lebecque S, Romero P, Pittet M, Rimoldi D, Liénard D, Gugerli O, Ferradini L, Robert C, Avril MF, Zitvogel L, Angevin E. · Immunology/Institut National de la Santé et de la Recherche Médicale ERM0208 and Immunotherapy Unit, Department of Pathology, Dermatology Unit, Institut Gustave-Roussy, 94805 Villejuif Cedex, France. · Cancer Res. · Pubmed #15026362 links to  free full text

Abstract: The clinical relevance of dendritic cells (DCs) at the tumor site remains a matter of debate concerning their role in the generation of effective antitumor immunity in human cancers. We performed a comprehensive immunohistochemical analysis using a panel of DC-specific antibodies on regressing tumor lesions and sentinel lymph nodes (SLNs) in melanoma patients. Here we show in a case report involving spontaneous regression of metastatic melanoma that the accumulation of DC-Lamp+ DCs, clustered with tumor cells and lymphocytes, is associated with local expansion of antigen-specific memory effector CTLs. These findings were extended in a series of 19 melanoma-positive SLNs and demonstrated a significant correlation between the density of DC-Lamp+ DC infiltrates in SLNs with the absence of metastasis in downstream lymph nodes. This study, albeit performed in a limited series of patients, points to a pivotal role of mature DCs in the local expansion of efficient antitumor T-cell-mediated immune responses at the initial sites of metastasis and may have important implications regarding the prognosis, staging, and immunotherapy of melanoma patients.

Valmori D, Dutoit V, Ayyoub M, Rimoldi D, Guillaume P, Liénard D, Lejeune F, Cerottini JC, Romero P, Speiser DE. · Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital (CHUV), 1011 Lausanne, Switzerland. · Cancer Immun. · Pubmed #14580186 links to  free full text

Abstract: The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

Karanikas V, Lurquin C, Colau D, van Baren N, De Smet C, Lethé B, Connerotte T, Corbière V, Demoitié MA, Liénard D, Dréno B, Velu T, Boon T, Coulie PG. · Cellular Genetics Unit, Institute of Cellular Pathology, Université de Louvain, Brussels, Belgium. · J Immunol. · Pubmed #14568971 links to  free full text

Abstract: We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained. Four patients who showed tumor regression were analyzed, and an anti-MAGE-3.A1 CTL response was observed in three of these patients. Postvaccination frequencies of anti-MAGE-3.A1 CTL were 3 x 10(-6), 3 x 10(-3), and 3 x 10(-7) of the blood CD8 T cells, respectively. These three responses were monoclonal. No anti-MAGE-1.A1 CTL response was observed. These results indicate that, like peptide immunization, ALVAC immunization produces monoclonal responses. They also suggest that low-level antivaccine CTL responses can initiate a tumor regression process. Taken together, our analysis of anti-MAGE-3.A1 T cell responses following peptide or ALVAC vaccination shows a degree of correlation between CTL response and tumor regression, but firm conclusions will require larger numbers.