Accuracy of sentinel lymph node dissection for melanoma staging in the presence of a collision tumour with a lymphoproliferative disease.

Sentinel lymph node dissection (SLND) identifies melanoma patients with metastatic disease who would benefit from radical lymph node dissection (RLND). Rarely, patients with melanoma have an underlying lymphoproliferative disease, and melanoma metastases might develop as collision tumours in the sentinel lymph node (SLN). The aim of this study was to measure the incidence and examine the effect of collision tumours on the accuracy of SLND and on the validity of staging in this setting. Between 1998 and 2012, 750 consecutive SLNDs were performed in melanoma patients using the triple technique (lymphoscintigraphy, gamma probe and blue dye). The validity of SLND in collision tumours was analysed. False negativity was reflected by the disease-free survival. The literature was reviewed on collision tumours in melanoma. Collision tumours of melanoma and chronic lymphocytic leukaemia (CLL) were found in two SLN and in one RLND (0.4%). Subsequent RLNDs of SLND-positive cases were negative for melanoma. The patient with negative SLND developed relapse after 28 months with an inguinal lymph node metastasis of melanoma; RLND showed collision tumours. The literature review identified 12 cases of collision tumours. CLL was associated with increased melanoma incidence and reduced overall survival. This is, to our knowledge, the first assessment of the clinical value of SLND when collision tumours of melanoma and CLL are found. In this small series of three patients with both malignancies present in the same lymph node basin, lymphocytic infiltration of the CLL did not alter radioisotope uptake into the SLN. No false-negative result was observed. Our data suggest the validity of SLND in collision tumours, but given the rarity of the problem, further studies are necessary to confirm this reliability.

Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood

Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.

Network-guided analysis of genes with altered somatic copy number and gene expression reveals pathways commonly perturbed in metastatic melanoma.

Cancer genomes frequently contain somatic copy number alterations (SCNA) that can significantly perturb the expression level of affected genes and thus disrupt pathways controlling normal growth. In melanoma, many studies have focussed on the copy number and gene expression levels of the BRAF, PTEN and MITF genes, but little has been done to identify new genes using these parameters at the genome-wide scale. Using karyotyping, SNP and CGH arrays, and RNA-seq, we have identified SCNA affecting gene expression ('SCNA-genes') in seven human metastatic melanoma cell lines. We showed that the combination of these techniques is useful to identify candidate genes potentially involved in tumorigenesis. Since few of these alterations were recurrent across our samples, we used a protein network-guided approach to determine whether any pathways were enriched in SCNA-genes in one or more samples. From this unbiased genome-wide analysis, we identified 28 significantly enriched pathway modules. Comparison with two large, independent melanoma SCNA datasets showed less than 10% overlap at the individual gene level, but network-guided analysis revealed 66% shared pathways, including all but three of the pathways identified in our data. Frequently altered pathways included WNT, cadherin signalling, angiogenesis and melanogenesis. Additionally, our results emphasize the potential of the EPHA3 and FRS2 gene products, involved in angiogenesis and migration, as possible therapeutic targets in melanoma. Our study demonstrates the utility of network-guided approaches, for both large and small datasets, to identify pathways recurrently perturbed in cancer.

Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.

Analysis of viral- and tumor antigen-specific CD4 T cell responses in healthy donors and melanoma patients

Résumé Des tentatives pour développer des traitements anti-cancéreux basés sur l'utilisation d'antigènes tumoraux ont commencé il y a plus de 10 ans. Depuis quelques années, un certain intérêt s'est portée sur une sous-population particulière des cellules du système immunitaire, les lymphocytes T CD4. Ces cellules jouent un rôle central dans les réponses immunitaires tant contre les virus que contre les cellules tumorales. Comme d'autres lymphocytes T, ces cellules sont activées de manière spécifique en reconnaissant un morceau d'antigène, appelé peptide. Ces peptides proviennent soit de protéines des cellules de l'hôte, soit des protéines étrangères (virus ou bactéries) soit de cellules transformées (cellules tumorales) et sont présentés aux lymphocytes T par des molécules du soi appelées CMH (complexe majeur d'histocompatibilité). Dans le cas des lymphocytes T CD4, ces molécules sont plus précisément des molécules du CMH de classe II (CMH II). Mis à part l'intérêt porté aux réponses médiées par les lymphocytes T cytotoxiques, un intérêt croissant pour les lymphocytes T CD4 s'est développé à cause de la place centrale qu'occupent ces cellules dans les réponses immunitaires. L'identification d'épitopes présentés par des molécules du CMH de classe II dérivés d'un grand nombre d'antigènes tumoraux, ainsi que le développement de techniques permettant de suivre les réponses immunitaires, offre des opportunités pour étudier de manière quantitative et qualitative les lymphocytes T CD4 spécifiques pour un antigène particulier chez des patients cancéreux. De plus, ces épitopes permettent d'induire des réponses médiées par les lymphocytes T CD4 et CD8 chez ces mêmes patients. Dans ce travail, notre premier but était de valider l'utilisation de multimères formés par des complexes peptide:molécules de CMH de class II (pCMH II) pour quantifier la réponse des cellules T CD4 dirigée contre l'épitope HA307-319 dérivé de la protéine hémaglutinine du virus de la grippe et présenté par HLA-DRB1*0401. En analysant des échantillons provenant de volontaires sains ayant reçus un vaccin contre la grippe, nous avons pu démontrer une expansion et une activation transitoires des lymphocytes T CD4 spécifiques pour le peptide HA307-319 après vaccination. De plus, les multimères pCMH II nous ont permis d'analyser plus en détails hétérogénéité des cellules T CD4 spécifiques pour le peptide HA307-319 présents dans le sang périphérique d'individus sains. Par la suite, notre but a été d'analyser les réponses des lymphocytes T CD4 spécifiques pour l'antigène Melan-A chez des patients atteints de mélanome métastatique. Nous avons tout d'abord démontré la présence de cellules T CD4 spécifiques pour l'épitope Melan-A51-73, présenté par HLA-DRBl*0401, qui avait déjà été préalablement décrit. Ensuite, nous avons décrit et caractérisé 2 nouveaux peptides issus de Melan-A qui sont présentés aux cellules T CD4 par différentes molécules du CMH de clans II. Des cellules spécifiques pour ces deux épitopes ont été trouvées chez 9/ 16 patients analysés. De plus, des multimères pCMH II chargés avec un des épitopes nous ont permis de détecter ex vivo des lymphocytes T CD4 spécifiques pour Melan-A dans le sang périphérique d'un patient atteint de mélanome. Mis ensemble, tous ces résultats suggèrent une potentielle utilisation des multimères pCMH II pour analyser en détail les lymphocytes T CD4 spécifiques d'antigènes définis. Cependant, le suivi ex vivo de telles cellules ne semble être possible que dans des cas bien particuliers. Néanmoins, les nouveaux épitopes issus de Melan-A et présentés par des molécules du CMH de classe II que nous avons décrits dans cette étude aideront à étudier plus en détails les lymphocytes T CD4 spécifiques pour Melan-A chez des patients atteints de mélanome, un sujet d'étude sur lequel peu de résultats sont à ce jour disponibles. Summary Attempts to develop cancer vaccines based on molecularly defined tumorassociated antigens were initiated more than 10 years ago. Apart from CTLmediated anti-tumor immunity, interests are. now focused on CD4 T cells that are central players of immune responses. The identification of MHC class-II-restricted epitopes from numerous tumor antigens together with the development of monitoring tools offers the opportunity to quantitatively and qualitatively study antigen-specific CD4 T lymphocytes in cancer patients and to induce both CTL and T helper responses in cancer patients. In this work, we first aimed at validating the use of peptide:MHC class II complex (pMHC II) multimers to quantitate the CD4 T cell response against the hemagglutinin-derived epitope HAso~-si9 from influenza virus presented by HLA-DRBl*0401. By analysing samples from healthy volunteers vaccinated with ananti-influenza vaccine, we could demonstrate a transient expansion and activation of HA-specific CD4 T cells after treatment. Moreover, pMHC II multimers helped us to study the heterogeneity of HAspecific CD4 T cells found in peripheral blood of healthy individuals. Then, we aimed to analyse Melan-A-specific CD4 T cell responses in metastatic melanoma patients. We first demonstrated the presence of CD4 T cells specific for the previously described Melan-A51_73 epitope presented by HLA-DRB 1 *0401 in peripheral blood of those patients. Second, we described and characterised 2 new Melan-A-derived peptides that are presented by different MHC II molecules to CD4 T cells. Specific cells for these epitopes were found in 9/ 16 rnelánoma patients analysed. In addition, pMHC II multimers loaded with one of the two epitopes allowed us to detect ex vivo Melan-A-specific CD4 T cells in peripheral blood of a melanoma patient. Together, these results suggest a potential use of pMHC II multimers in analysing in detail antigen-specific CD4 T cells. However, ex vivo monitoring of such cells will be possible only in particular conditions. Nevertheless, the new Melan-A-derived MHC II-restricted epitopes described here will help to study in more detail Melan-A-specific CD4 T cells in melanoma patients, a field where only scarce data are available.

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.

Expression of somatostatin receptors in human melanoma cell lines: effect of two different somatostatin analogues, octreotide and SOM230, on cell proliferation

Somatostatin analogues (SAs) are potential anticancer agents. This study was designed to investigate the expression of somatostatin receptors (SSTRs) in melanoma cells and the effect of two SAs on cell proliferation and viability. Eighteen primary and metastatic human cutaneous melanoma cell lines were treated with octreotide and SOM230. Expression of SSTR1, SSTR2, SSTR3 and SSTR5 was assessed by real-time polymerase chain reaction. Proliferation, viability and cell death were assessed using standard assays. Inhibition was modelled by mixed-effect regression. Melanoma cells expressed one or more SSTR. Both SAs inhibited proliferation of most melanoma cell lines, but inhibition was less than 50%. Neither SA affected cell viability or induced cell death. The results suggest that melanoma cell lines express SSTRs. The SAs investigated, under the conditions used in this study, did not, however, significantly inhibit melanoma growth or induce cell death. Novel SAs, combination therapy with SAs and their anti-angiogenic properties should be further investigated.

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.

Cytoplasmic accumulation of NCoR in malignant melanoma: Consequences of altered gene repression and prognostic significance

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy available in advanced stages. Nuclear corepressor (NCoR) is an essential regulator of gene transcription, and its function has been found deregulated in different types of cancer. In colorectal cancer cells, loss of nuclear NCoR is induced by Inhibitor of kappa B kinase (IKK) through the phosphorylation of specific serine residues. We here investigate whether NCoR function impacts in MM, which might have important diagnostic and prognostic significance. By IHC, we here determined the subcellular distribution of NCoR in a cohort of 63 primary invasive MM samples, and analyzed its possible correlation with specific clinical parameters. We therefore used a microarray-based strategy to determine global gene expression differences in samples with similar tumor stage, which differ in the presence of cytoplasmic or nuclear NCoR. We found that loss of nuclear NCoR results in upregulation of a specific cancer-related genetic signature, and is significantly associated with MM progression. Inhibition of IKK activity in melanoma cells reverts NCoR nuclear distribution and specific NCoR-regulated gene transcription. Analysis of public database demonstrated that inactivating NCoR mutations are highly prevalent in MM, showing features of driver oncogene.

Collagen binding integrins and cancer testis antigens in prostate cancer and melanoma

Camilla Pelo Collagen Binding Integrins and Cancer Testis Antigens in Prostate Cancer and Melanoma Department of Biochemistry, MediCity Research Laboratory, University of Turku, Finland Annales Universitatis Turkuensis, Painosalama Oy, Turku, Finland 2016 ABSTRACT Prostate cancer is the second most common cancer in men worldwide. The incidence of melanoma, in turn, is increasing faster than any other cancer incidences. In Finland, more than 5000 prostate cancer and 1200 new melanoma cases are diagnosed each year. One approach to further understand the cellular processes involved in prostate cancer and melanoma is to gain better knowledge about alterations in gene expression and their potential impact on the progression of the diseases. This thesis is focused on expression studies in two gene families; integrins and cancer testis antigens (CT antigens), in human prostate adenocarcinoma and advanced human melanoma. Integrins are heterodimeric transmembrane receptors which regulate many important cellular processes such as cell proliferation, migration and survival. CT antigens are frequently expressed in different types of cancers, but are only expressed in testis in healthy individuals. CT antigens are also highly immunogenic proteins. Due to the properties mentioned above, integrins and CT antigens can function as target molecules for the development of cancer diagnostics and drugs. One of the main purposes of this thesis was to study the expression of the four collagen binding integrins α1β1, α2β1, α10β1, α11β1 and the cancer testis antigen 16 (CT16) in cancer cell lines and human tissues of prostate cancer and metastatic melanoma. Additional aims included studies on the biological role of CT16 and the abundance of CT16 in sera of advanced melanoma patients. The prognostic and diagnostic significance of CT16 and the collagen binding integrins were also evaluated. Expression studies on collagen binding integrins and the CT antigen CT16 in melanoma and prostate cancer were limited and the biological role of CT16 was unknown. In this thesis, the expression levels of α2β1 and α11β1 were found to be significantly altered in prostate cancer tissues. Integrin α2β1 decreased gradually during disease progression while α11 was elevated in prostate carcinoma compared to healthy tissues. In advanced melanoma, enhanced levels of α2 were associated with a significant shorter overall survival in advanced melanoma. In this thesis, CT16 was identified as a frequently expressed melanoma CT antigen with an anti-apoptotic function. To conclude, this thesis presents α2β1 and CT16, as potential and promising biomarkers for advanced melanoma. This thesis reports also the first functional study of CT16. Keywords: Collagen binding integrins, α1β1, α2β1, α10β1, α11β1, Cancer Testis antigens, CT16, melanoma, prostate cancer, expression

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on B-10 distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The B-10 concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the B-10 concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of B-10 uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Recent skin self-examination and doctor visits in relation to melanoma risk and tumour depth

BACKGROUND Little is known about the potential benefit of skin self-examination for melanoma prevention and early detection. Objectives: To determine whether skin self-examination is associated with reduced melanoma risk, self-detection of tumours, and reduced risk of deeper melanomas. METHODS We used data from a population-based case-control study (423 cases, 678 controls) to assess recent skin self-examination in relation to self-detection, melanoma risk and tumour depth ( ≤1 mm; > 1 mm). Logistic regression was used to estimate odds ratios (ORs) and confidence intervals (CIs) for associations of interest. RESULTS Skin self-examination conducted 1-11 times during a recent year was associated with a possible decrease in melanoma risk (OR 0·74; 95% CI 0·54-1·02). Melanoma risk was decreased for those who conducted skin self-examination and saw a doctor (OR 0·52; 95% CI 0·30-0·90). Among cases, those who examined their skin were twice as likely to self-detect the melanoma (OR 2·23; 95% CI 1·47-3·38), but self-detection was not associated with shallower tumours. Tumour depth was reduced for those who conducted skin self-examination 1-11 times during a recent year (OR 0·39; 95% CI 0·18-0·81), but was not influenced by seeing a doctor, or by conducting skin self-examination and seeing a doctor. CONCLUSIONS Risk of a deeper tumour and possibly risk of melanoma were reduced by skin self-examination 1-11 times annually. Melanoma risk was markedly reduced by skin self-examination coupled with a doctor visit. We cannot, however, exclude the possibility that our findings reflect bias or confounding. Additional studies are needed to elucidate the potential benefits of skin self-examination for melanoma prevention and early detection.

CHARACTERIZATION OF DIFFERENTIATION AND PROGNOSTIC BIOMARKERS ON CD8+ TUMOR-INFILTRATING LYMPHOCYTES IN METASTATIC MELANOMA

CD8+ cytotoxic T lymphocytes (CTL) frequently infiltrate tumors, yet most melanoma patients fail to undergo tumor regression. We studied the differentiation of the CD8+ tumor-infiltrating lymphocytes (TIL) from 44 metastatic melanoma patients using known T-cell differentiation markers. We also compared CD8+ TIL against the T cells from matched melanoma patients’ peripheral blood. We discovered a novel subset of CD8+ TIL co-expressing early-differentiation markers, CD27, CD28, and a late/senescent CTL differentiation marker, CD57. This CD8+CD57+ TIL expressed a cytolytic enzyme, granzyme B (GB), yet did not express another cytolytic pore-forming molecule, perforin (Perf). In contrast, the CD8+CD57+ T cells in the periphery were CD27-CD28-, and GBHi and PerfHi. We found this TIL subset was not senescent and could be induced to proliferate and differentiate into CD27-CD57+, perforinHi, mature CTL. This further differentiation was arrested by TGF-β1, an immunosuppressive cytokine known to be produced by many different kinds of tumors. Therefore, we have identified a novel subset of incompletely differentiated CD8+ TIL that resembled those found in patients with uncontrolled chronic viral infections. In a related study, we explored prognostic biomarkers in metastatic melanoma patients treated in a Phase II Adoptive Cell Therapy (ACT) trial, in which autologous TIL were expanded ex vivo with IL-2 and infused into lymphodepleted patients. We unexpectedly found a significant positive clinical association with the infused CD8+ TIL expressing B- and T- lymphocyte attentuator (BTLA), an inhibitory T-cell receptor. We found that CD8+BTLA+ TIL had a superior proliferative response to IL-2, and were more capable of autocrine IL-2 production in response to TCR stimulation compared to the CD8+BTLA- TIL. The CD8+BTLA+ TIL were less differentiated and resembled the incompletely differentiated CD8+ TIL described above. In contrast, CD8+BTLA- TIL were poorly proliferative, expressed CD45RA and killer-cell immunoglobulin-like receptors (KIRs), and exhibited a gene expression signature of T cell deletion. Surprisingly, ligation of BTLA by its cognate receptor, HVEM, enhanced the survival of CD8+BTLA+ TIL by activating Akt/PKB. Our studies provide a comprehensive characterization of CD8+ TIL differentiation in melanoma, and revealed BTLA as a novel T-cell differentiation marker along with its role in promoting T cell survival.