A novel population of human melanoma-specific CD8 T cells recognizes Melan-AMART-1 immunodominant nonapeptide but not the corresponding decapeptide.

HLA-A2-restricted cytolytic T cells specific for the immunodominant human tumor Ag Melan-A(MART-1) can kill most HLA-matched melanoma cells, through recognition of two naturally occurring antigenic variants, i.e., Melan-A nonamer AAGIGILTV and decamer EAAGIGILTV peptides. Several previous studies have suggested a high degree of TCR cross-reactivity to the two peptides. In this study, we describe for the first time that some T cell clones are exclusively nonamer specific, because they are not labeled by A2/decamer-tetramers and do not recognize the decamer when presented endogenously. Functional assays with peptides gave misleading results, possibly because decamers were cleaved by exopeptidases. Interestingly, nonapeptide-specific T cell clones were rarely Valpha2.1 positive (only 1 of 19 clones), in contrast to the known strong bias for Valpha2.1-positive TCRs found in decamer-specific clones (59 of 69 clones). Molecular modeling revealed that nonapeptide-specific TCRs formed unfavorable interactions with the decapeptide, whereas decapeptide-specific TCRs productively created a hydrogen bond between CDR1alpha and glutamic acid (E) of the decapeptide. Ex vivo analysis of T cells from melanoma metastases demonstrated that both nonamer and decamer-specific T cells were enriched to substantial frequencies in vivo, and representative clones showed efficient tumor cell recognition and killing. We conclude that the two peptides should be regarded as distinct epitopes when analyzing tumor immunity and developing immunotherapy against melanoma.

PD-1 and Tim-3 regulate the expansion of tumor antigen-specific CD8⁺ T cells induced by melanoma vaccines.

Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma.

Phenotyping of 60 cultured human gliomas and 34 other neuroectodermal tumors by means of monoclonal antibodies against glioma, melanoma and HLA-DR antigens.

The reactivity spectrum of three monoclonal antibodies (Mabs) to human malignant glioma, five Mabs to melanomas and one Mab anti-HLA-DR was investigated by an indirect antibody binding radioimmunoassay on a panel of cells derived from 60 glioma lines, including 47 malignant astrocytomas, 11 low-grade astrocytomas and two malignant ependymomas as well on cells from 12 melanoma, three neuroblastoma, three medulloblastoma, two schwannoma, two retinoblastoma, two choroïd plexus papilloma, ten meningioma and 12 unrelated tumor lines. The anti-glioma Mabs BF7 and GE2 reacted preferentially with gliomas, while the anti-glioma Mab CG12 reacted with gliomas, melanomas, neuroblastomas and medulloblastomas. The five anti-melanoma Mabs reacted with gliomas, neuroblastomas and medulloblastomas. The anti-HLA-DR Mab D1-12 reacted with gliomas, melanomas and some meningiomas. On the basis of the data presented, we describe three different antigenic systems; the first one is glioma-associated, the second one is related to differentiation antigens expressed on cells derived from the neuroectoderm and the third is represented by HLA-DR antigens which are expressed not only on B-lymphoblastoid cells but also on melanomas and gliomas.

In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.

Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells.

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.

The human T cell response to melanoma antigens.

The cornerstone of the concept of immunosurveillance in cancer should be the experimental demonstration of immune responses able to alter the course of in vivo spontaneous tumor progression. Elegant genetic manipulation of the mouse immune system has proved this tenet. In parallel, progress in understanding human T cell mediated immunity has allowed to document the existence in cancer patients of naturally acquired T cell responses to molecularly defined tumor antigens. Various attributes of cutaneous melanoma tumors, notably their adaptability to in vitro tissue culture conditions, have contributed to convert this tumor in the prototype for studies of human antitumor immune responses. As a consequence, the first human cytolytic T lymphocyte (CTL)-defined tumor antigen and numerous others have been identified using lymphocyte material from patients bearing this tumor, detailed analyses of specific T cell responses have been reported and a relatively large number of clinical trials of vaccination have been performed in the last 15 years. Thus, the "melanoma model" continues to provide valuable insights to guide the development of clinically effective cancer therapies based on the recruitment of the immune system. This chapter reviews recent knowledge on human CD8 and CD4 T cell responses to melanoma antigens.

CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.

Human CD8 T-cell responses to Epstein-Barr virus and Cytomegalovirus in healthy donors and melanoma patients

Summary The mechanisms regulating the protective immune T-cell responses generated against the persistent Epstein-Barr virus (EBV) and Cytomegaloviru_s (CNIV) remain poorly understood. We analyzed the dynamics of cellular differentiation and T-cell receptor (TCR) clonotype selection of EBV- and CMV-specific T-cells in healthy adults and melanoma patients. While these responses could be subdivided into four T lymphocyte populations, théir proportions varied between EBV and CMV specific responses. Phenotypic and TCR clonotypic analyses supported a linear model of differentiation from the early-differentiated (EM/CD28pos) subset to the late-differentiatdc (EMRA/CD28neg) subset. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28neg subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, as some clonotypes were selected with differentiation, while others were not. Latedifferentiated CMV-specific clonotypes were mostly characterized by TCRs with lower dependency on CD8 co-receptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of four years. This work was extended to the study of EBV-specific CD8 T-cell responses in melanoma patients undergoing transient lymphodepletion, followed by adoptive cell transfer (ACT) and immune reconstitution for thè treatment of their tumors. Following treatment regimen, we first observed an increase in the proportion of virus-specific T-cells in 3 out of 5 patients, accompanied by a more differentiated phenotype (EMRA/CD28neg), compared to specific cells of healthy individuals. Yet, similarly to healthy donors, clonotype selection and composition of virus-specific T-cells varied along the pathway of cellular differentiation, with some clonotypes being selected with differentiation, while others were not. Intriguingly, no novel clonotypes emerged following transient immuno-suppression and homeostatic proliferation, finding which was subsequently explained by the absence of EBV reactivation. The distribution of each clonotype within early- and late-differentiated T-cell subsets in 4 out 5 patients was highly stable over time, with those clonotypes initially found before the start of treatment that were again present at specific differentiation stages after transient lymphodepletion and ACT. These findings uncover novel features of the highly sophisticated control of steady state protective T-cell immune responses against persistent herpesviruses in healthy adults. Furthermore they reveal the striking stability of these responses in terms of clonotype selection and composition with T-cell differentiation even in situations where the immune system has been. challenged. Résumé : Les mécanismes qui régulent les réponses immunitaires de type protectrices, générées contre les virus chroniquement persistants tels que l'Epstein-Barr (EBV) ou le Cytomegalo (CMV) restent largement inconnus. Nous avons analysé la différenciation des lymphocytes T spécifiques pour ces virus, ainsi que la composition des clonotypes T (par leur récepteur T) chez les donneurs sains. Les réponses immunes peuvent être classifiées en quatre souspopulations majeures de lymphocytes T, cependant, leur proportion varie entre les réponses spécifiques contre EBV ou CMV. Ces analyses soutiennent le modèle linéaire de différenciation, à partir de la population non différenciée (EM/CD28pos) vers la population plus différenciée (ENIIZA/CD28neg). De plus, nos données sur la composition clonale de ces cellules T spécifiques ont révélé des répertoires TCR restreints, pour la réponse anti-CMV, et relativement diversifiés contre EBV. Tous les clonotypes spécifiques de ces virus identifiés dans la sous-population différenciée EMRA/CD28neg, ont également été retrouvés dans la population de cellules "mémoires". Toutefois, de fortes différences ont été observées dans les schémas de domination de ces sous-populations, en effet, certains clonotypes étaient sélectionnés avec la différenciation, alors que d'autres ne l'étaient pas. Nous avons également démontré que ces clonotypes différenciés et spécifiques pour le CMV sont caractérisés par des TCRs à faible dépendance en regard de la coopération du corécepteur CD8. Néanmoins, tous les clonotypes affichent une avidité fonctionnelle similaire, suggérant un rôle compensatoire du CD8, dans le cas des clonotypes avec une faible avidité du TCR En définitive, la composition et la sélection des clonotypes spécifiques pour chaque virus et pour chaque sous-population suit un schéma de différenciation hautement conservé au cours du temps, avec la présence de ces mêmes clonotypes au même stade de différenciation sur une période de quatre ans. Ce travail a été étendu à l'étude des réponses T CD8+ spécifiques pour le virus EBV chez les patients atteints de mélanome et recevant dans le cadre du traitement de leurs tumeurs une lymphodéplétion transitoire, suivie d'un transfert adoptif de cellules et d'une reconstitution immunitaire. Au cours de cette thérapie, nous avons en premier lieu observé pour 3 des 5 patients une augmentation de la proportion de cellules T spécifiques pour le virus, accompagné d'un phénotype plus différencié (EMRA/CD28neg), et ceci comparativement à des cellules spécifiques d'individus sains. Pourtant, comme nous l'avons observé chez les donneurs sains, la sélection et la composition des clonotypes T spécifiques varient tout au long de la différenciation cellulaire, avec certains clonotypes sélectionnés et d'autres qui ne le sont pas. Étonnamment, aucun nouveau clonotype n'a émergé après l'immuno-suppression transitoire et la prolifération homéostatique. Cette observation trouve son explication par une absence de réactivation du virus EBV chez ces patients, et ce malgré leur traitement. De plus, la distribution de chaque clonotype parmi ces sous-populations non-différenciées et différenciées reste stable au cours du traitement. Ainsi, les mêmes clonotypes initialement identifiés avant le début du traitement sont présents aux mêmes stades de différenciation après la lymphodéplétion et la prolifération homéostatique. Ces résultats ont permis d'identifier de nouveaux mécanismes impliqués dans la régulation hautement «sophistiquée » des réponses immunitaires T contre les virus persistants EBV et CMV chez les donneurs sains. En particulier, ils révèlent la grande stabilité de ces réponses en termes de sélection et de composition des clonotypes avec la différenciation cellulaire, et ce dans les situations chroniques, ainsi que dans les situations dans lesquelles le système immunitaire a été profondément perturbé.

The curcumin analog DM-1 induces apoptotic cell death in melanoma

The main difficulty in the successful treatment of metastatic melanoma is that this type of cancer is known to be resistant to chemotherapy. Chemotherapy remains the treatment of choice, and dacarbazine (DTIC) is the best standard treatment. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and antimetastatic properties. The objective of this study was to evaluate the signaling pathways involved in melanoma cell death after treatment with DM-1 compared to the standard agent for melanoma treatment, DTIC. Cell death was evaluated by flow cytometry for annexin V and iodide propide, cleaved caspase 8, and TNF-R1 expression. Hoechst 33342 staining was evaluated by fluorescent microscopy; lipid peroxidation and cell viability (MTT) were evaluated by colorimetric assays. The antiproliferative effects of the drugs were evaluated by flow cytometry for cyclin D1 and Ki67 expression. Mice bearing B16F10 melanoma were treated with DTIC, DM-1, or both therapies. DM-1 induced significant apoptosis as indicated by the presence of cleaved caspase 8 and an increase in TNF-R1 expression in melanoma cells. Furthermore, DM-1 had antiproliferative effects in this the same cell line. DTIC caused cell death primarily by necrosis, and a smaller melanoma cell population underwent apoptosis. DTIC induced oxidative stress and several physiological changes in normal melanocytes, whereas DM-1 did not significantly affect the normal cells. DM-1 antitumor therapy in vivo showed tumor burden decrease with DM-1 monotherapy or in combination with DTIC, besides survival rate increase. Altogether, these data confirm DM-1 as a chemotherapeutic agent with effective tumor control properties and a lower incidence of side effects in normal cells compared to DTIC.

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

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.

Breast cancer suppressor candidate-1 (BCSC-1) is a melanoma tumor suppressor that down regulates MITF.

Understanding the molecular aberrations involved in the development and progression of metastatic melanoma (MM) is essential for a better diagnosis and targeted therapy. We identified breast cancer suppressor candidate-1 (BCSC-1) as a novel tumor suppressor in melanoma. BCSC-1 expression is decreased in human MM, and its ectopic expression in MM-derived cell lines blocks tumor formation in vivo and melanoma cell proliferation in vitro while increasing cell migration. We demonstrate that BCSC-1 binds to Sox10, which down regulates MITF, and results in a switch of melanoma cells from a proliferative to a migratory phenotype. In conclusion, we have identified BCSC-1 as a tumor suppressor in melanoma and as a novel regulator of the MITF pathway.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

The prognostic value of minimally involved melanoma sentinel lymph nodes

Introduction: la biopsie du ganglion sentinelle (GS) est une procédure reconnue et fiable pour établir le stade ganglionnaire du mélanome cutané. Le GS est le facteur pronostique le plus puissant pour la survie des patients atteints d'un mélanome à risque intermédiaire, cliniquement localisé. Celui-ci est métastatique dans environ 15-30% des cas. Lorsque le GS est positif, un curage de l'aire ganglionnaire concernée est généralement entrepris. Néanmoins, seuls 20-25% de ces patients présentent des ganglions non-sentinelles (GNS) métastatiques. Ces données suggèrent que le curage, et les risques opératoires qui y sont associés, n'est peut-être pas nécessaire chez le trois-quarts de ces patients. Un autre aspect est que l'impact sur la survie des curages basé sur le résultat du GS n'est pas clairement démontré. La nécessité de ce curage d'emblé est actuellement en cours d'évaluation par un protocole international (Multicenter Selective Lymphadenectomy Trial II : MSLT II). Plusieurs auteurs ont essayé de classifier la charge tumorale du GS afin d'évaluer s'il était possible d'épargner le curage à certains patients et de mieux affiner ce facteur pronostic sans succès. En 2009, le Groupe Mélanome de l'EORTC (European Organisation for Research and Treatment of Cancer) a recommandé un protocole d'évaluation anatomopathologique du GS-positif en trois items: (1) la localisation micro-anatomique des métastases à l'intérieur du ganglion selon Dewar (A = sous-capsulaire, B = combinée sous-capsulaire and parenchymateuse, C = parenchymateuse, D = multifocale, and'E = extensive) ; (2) la mesure de la taille tumorale dans le ganglion selon les critères de Rotterdam pour le diamètre maximal. Le diamètre de la plus grande métastase est exprimé en nombre absolu et (3) la taille tumorale stratifiée par catégories : <0.1mm, 0.1-1.0mm et >1.0 mm. Le but de cette étude rétrospective d'une cohorte de patients, était d'investiguer les résultats des GS-positifs et d'analyser les facteurs pronostiques de la survie à la lumière des recommandations de l'EORTC. Ainsi que de comparer les sous-groupes du GS-positif avec une invasion minimale (taille tumorale <0.1mm et/ou atteinte sous-capsulaire) avec le GS-négatif. Les facteurs pouvant prédire la présence de GNS- positif ont également été analysés. Matériel et méthode : une étude des dossiers a été réalisée pour les 499 patients consécutifs entre 1997 et 2008 qui ont eu une biopsie du GS dans notre institution. Le dégrée d'envahissement du GS-positif a été entièrement revue par l'équipe référente de l'Institut de Pathologie (Dresse E. Saiji et Dresse H. Bouzourène) selon les recommandations de l'EORTC. Des analyses univariées et multivariées des potentiels facteuis pronostics ont été réalisées. Des analyses de survie ont également été effectuées avec des courbes d'estimation de Kaplan-Meier combinées à une régression de Cox. Le protocole a été accepté par la Commission d'Ethique. Résultats: un GS-positif a été trouvé chez 123 (25%) patients panni les 499 qui ont bénéficié d'une biopsie. Avec un suivi médian de 52 mois, la survie à 5 ans sans récidive (SSR), spécifique à la maladie (SS) et globale (SG) étaient de 88%, 94%, et 90% respectivement pour les patients avec GS-négatif. Concernant les GS avec invasion minimale, 21 patients étaient dans le sous-groupe <0.1 mm selon les critères de Rotterdam et 52 patients dans le sous-groupe sous-capsulaire selon Dewar. La survie dans ces deux sous-groupes était de 80% et 57% pour la SSR, 87% et 70% pour la SS, 87% et 68% pour la SG, respectivement. L'analyse multivariée des GS-positifs a montré que les facteuis suivants influençaient significativement la survie (SSR, SS et SG): l'épaisseur selon Breslow de la tumeur primaire (p=0.002, 0.006, 0.004), la taille tumorale du GS-positif >0.1 mm (p= 0.01, 0.04, 0.03), le genre masculin (p=0.06, 0.005, 0.002) et l'ulcération de la tumeur primaire (p=0.05, 0.03, 0.007). L'analyse des sous-groupes avec invasion minimale n'a pas permis d'établir de facteur pour prédire la négativité des GNSs. Conclusion: La classification du GS-positif par la taille tumorale selon les critères de Rotterdam est un facteur pronostique simple et utile pour évaluer la survie des patients atteints de mélanome. Nous avons observé une tendance (non statistiquement significative) d'une survie diminuée pour le sous-groupe des patients avec GS-positif et une taille de la métastase <0.1 mm comparée à celle des patients avec GS-négatif. Ceci nous incite à conclure que ce sous-groupe de patients ne devrait pas être assimilé et traité comme ceux qui ont un GS-négatif. D'autre part nos résultats montrent que la localisation micro-anatomique selon Dewar n'est pas un outil pronostique utile pour évaluer la survie, ni pour prédire le status des GNSs.

Frequent cytolytic T-cell responses to peptide MAGE-A10(254-262) in melanoma.

MAGE genes encode tumor-specific shared antigens that are among the most interesting candidates for cancer vaccines. Despite extensive studies, however, CD8+ T-cell responses to MAGE-derived epitopes have been detected only occasionally in cancer patients, even after vaccination. In contrast with these findings, we report here that HLA-A2 melanoma patients respond frequently to the recently identified peptide MAGE-A10(254-262). Indeed, as assessed by staining with fluorescent HLA-A2/peptide MAGE-A10(254-262) tetramers, CD8+ T cells directed against this peptide were readily detectable in a large proportion of HLA-A2+ melanoma patients. These results provide new insight into the immunogenicity of MAGE antigens and underline the potential usefulness of MAGE-A10 peptide-based cancer vaccines.