Vaccine strategies against melanoma

Melanoma incidence increases and conventional antitumor therapies are often ineffective, encouraging the design of novel therapies. Several lines of evidence support the notion of an immunological control of melanoma growth. Based on this information, active immunotherapy (vaccination) and adoptive immunotherapy trials (T cell therapy) were conducted in metastatic melanoma patients. The proof of principle of effective immunotherapy was brought up by pionnering trials using tumor infiltrated lymphocytes in lymphodepleted recipients or anti-CTLA4 Ab leading to tumor eradication but also autoimmune diseases. With the identification and characterization of tumor antigens recognized by cytotoxic T lymphocytes, the utilization of tumor rejection antigens along with adjuvants become available as tumor vaccines. The last five years have witnessed the emergence of dendritic cell based-vaccines that were efficient in priming and/or boosting T cell responses in normal volunteers and patients. This review highlights preclinical bases of cancer vaccines, their clinical development and discusses their limits. Correlations between immunomonitoring and tumor regressions await larger trials.     

Lymphocyte subsets as prognostic markers for cancer patients receiving immunomodulative therapy

Immunogenic features of some malignancies have aroused interest in immunotherapy of cancer. Immunotherapy seems most effective in patients with a small tumour burden, and the focus of immunotherapy trials has, thus, lately been on adjuvant treatment. To enable further development of immunotherapy we need to know more about the mechanisms involved in host defence, especially when the system is influenced by extrinsic factors, that is, immunomodulative agents. T lymphocytes play an important role in the host defence against tumour cells trying to escape from immune surveillance. The mechanisms that regulate the host defence systems are complex, and the influence of extrinsic factors such as immunotherapeutic agents is poorly understood. Most data on lymphocyte subsets in malignant disease originate from melanoma or renal cell carcinoma (RCC) studies, although there are scattered data on lymphocyte subsets also in other malignancies. There are several studies implying that the relative amount of CD4+, CD8+, and natural killer (NK) cells may be important and that, by reducing the tumour burden or by using different therapeutic agents, we can stimulate the host defence. However, only some of these studies imply that these changes can have an impact on clinical outcome and prognosis. The findings of the studies reviewed in this paper are mostly encouraging, but whether the lymphocyte subsets have any value as prognostic markers in patients with malignancies receiving immunotherapy is still unclear. Large randomized immunotherapy trials including an observation arm give an ideal opportunity to recognize those immunological changes that are due to therapy, related to the natural host defence, or whether they have any prognostic value.     

Identification of differentially expressed genes in spontaneously regressing melanoma using the MeLiM swine model

Partial and some few cases of complete spontaneous regression have been observed in cutaneous melanoma patients but little is known about the molecular mechanisms involved. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a suitable animal model to study the phenomenon of spontaneous regression because MeLiM pigs exhibit naturally occurring melanomas which regress completely 6 months after birth. In this study, we used suppression subtractive hybridization (SSH) to identify molecular determinants of melanoma regression within swine melanoma tissues and melanoma cell cultures. Several markers involved in cell-adhesion, -communication, -motility, signal transduction, negative regulation of cell proliferation, transport and immune response were identified that correlated with melanoma regression whereas the main genes involved in melanin synthesis showed a strong downregulation. For the most differentially expressed genes, we validated the results obtained by SSH with qRT-PCR and with immunohistochemistry for some of them (CD9, MITF, RARRES1). Most notable, for the first time in melanoma, we identified the retinoic acid responder 1 gene (RARRES1) as a main actor of the regression process in melanoma. This first gene expression study in swine melanoma regression, may contribute to the finding of new therapeutic targets for human melanoma treatment.     

Expression profile of SIRT2 in human melanoma and implications for sirtuin-based chemotherapy

Melanoma is cancer of melanin-containing melanocyte cells. This neoplasm is one of the most deadly forms of skin cancer, and currently available therapeutic options are insufficient in significantly improve outcomes for many patients. Therefore, novel targets are required to effectively manage this neoplasm. Several sirtuins have previously been found to be upregulated in melanoma, so in this study, the expression profile of SIRT2 was determined. Employing a tissue microarray containing benign nevi, primary melanomas, and lymph node metastases, we have found that the tissue from lymph node metastases appears to have a significant upregulation of SIRT2 relative to primary tumors across the nuclear, cytoplasmic, and whole cell data. Additionally, SIRT2 staining was found to be higher in the nucleus of metastatic melanomas compared to cytoplasmic staining. As SIRT2 is considered to be a predominantly cytoplasmic protein, this is a novel and very interesting finding. This, combined with previous studies that show other sirtuins are increased in melanoma and involved in cellular proliferation and survival, leads to the suggestion that exploring pan-sirtuin inhibitors may be the best target for the next iteration of melanoma chemotherapeutics.     

Response of Solanum tuberosum to Myzus persicae infestation at different stages of foliage maturity

Young leaves of the potato Solanum tuberosum L. cultivar Kardal contain resistance factors to the green peach aphid Myzuspersicae （Sulzer） （Hemiptera： Aphididae） and normal probing behavior is impeded. However, M. persicae can survive and reproduce on mature and senescent leaves of the cv. Kardal plant without problems. We compared the settling ofM. persicae on young and old leaves and analyzed the impact of aphids settling on the plant in terms of gene expression. Settling, as measured by aphid numbers staying on young or old leaves, showed that after 21 h significantly fewer aphids were found on the young leaves. At earlier time points there were no difference between young and old leaves, suggesting that the young leaf resistance factors are not located at the surface level but deeper in the tissue. Gene expression was measured in plants at 96 h postinfestation, which is at a late stage in the interaction and in compatible interactions this is long enough for host plant acceptance to occur. In old leaves of cv. Kardal （compatible interaction）, M. persicae infestation elicited a higher number of differentially regulated genes than in young leaves. The plant response to aphid infestation included a larger number of genes induced than repressed, and the proportion of induced versus repressed genes was larger in young than in old leaves. Several genes changing expression seem to be involved in changing the metabolic state of the leaf from source to sink.     

Circulating gammadelta T cells in young/adult and old patients with cutaneous primary melanoma

BACKGROUND: In a previous study we demonstrated the existence of numerical and functional alterations of gammadelta T cells in healthy elderly. Recently, we analysed the involvement of gammadelta T lymphocytes in malignant melanoma, describing a lower frequency of circulating gammadelta T cells, an altered pattern of cytokine production, and an impaired in vitro expansion of these cells in primary cutaneous melanoma patients. METHODS: In this study we investigated the existence of numerical and functional alterations of circulating gammadelta T cells in young/adult and old melanoma patients, comparing the data obtained with age-matched healthy subjects. RESULTS: We demonstrated that the number of circulating gammadelta+ T cells was significantly and similarly reduced in young/adult and old melanoma patients and in old healthy subjects in comparison with young healthy donors. The decrease was due to a reduction of Vdelta2 T cells whereas the number of Vdelta1 T cells was not affected. A higher percentage of gammadelta+ T cells producing TNF-alpha was found in old healthy donors, whereas a reduced number of TNF-alpha-producing gammadelta+ T cells was present in melanoma patients independently by age. No significant difference was observed in IFN-gamma production. After a 10-day in vitro culture, both the percentage and the expansion index of gammadelta T cells, and in particular of Vdelta2 subset, were significantly and similarly reduced both in young/adult and old melanoma patients, and in healthy aged people, in comparison with young/adult healthy subjects. CONCLUSIONS: Our study demonstrates that the numerical and functional impairment of gammadelta T cells found in melanoma patients is not correlated with age and that it has characteristics very similar to the alterations of gammadelta T cells found in old healthy subjects. We suggest that a similar impairment of gammadelta T cell population may be related to the increased susceptibility to tumors present in the elderly as well as in the pathogenesis of malignant melanoma.     

Cloning and in vitro expression of a melanoma-associated antigen immunogenic in patients with melanoma

The purpose of this study was to identify human melanoma-associated Ag (MAA) that are immunogenic in patients, because these molecules may be useful immunogens to implement active specific immunotherapy. To this end, an expression cDNA library constructed from the human melanoma cell line A375 was screened with sera from patients with melanoma. A 1029-bp cDNA (designated D-1) was isolated. Its nucleotide sequence showed no significant homology with viral and mammalian sequences stored in GE-NETYX. cDNA D-1 hybridized to a 2.0-kb mRNA species from human melanoma, neuroblastoma, erythroleukemia, B lymphoid, and T lymphoid cell lines but not from a renal carcinoma cell line, PBL, and cultured skin fibroblasts. The D-1 clone produced a fusion protein that displayed a significantly higher reactivity with sera from patients with melanoma than from healthy controls. Furthermore, D-1 fusion protein induced in mice antibodies that immunoprecipitated a 50-kDa component from cultured human melanoma cells. The structural properties of D-1 MAA are different from those of previously described MAA. These results suggest that the approach we have applied may be useful to identify novel MAA expressed by melanoma cells. Furthermore, the immunogenicity of recombinant D-1 protein suggests that it may be a valuable immunogen to implement active specific immunotherapy in patients with melanoma, if additional experiments show that it has the appropriate tissue distribution.     

RNA editing signatures identify melanoma patients who respond to Pembrolizumab or Nivolumab treatment

Immunotherapy has improved the prognosis for many melanoma patients; however, our capacity to predict patient responses and to understand the biological differences between patients who will or will not respond is limited. Gene expression profiling of tumors from patients who respond to immunotherapy has focused on deriving primarily immune-related signatures; however, these have shown limited predictive power. Recent studies have highlighted the role of RNA editing in modulating resistance to immunotherapy. To evaluate the utility of RNA editing activity as a discriminative tool in predicting immunotherapy response, we conducted a retrospective analysis of RNA-sequencing data from melanoma patients treated with Pembrolizumab or Nivolumab. Here, we developed RNA editing signatures that can identify patients who will respond to immunotherapy with very high accuracy and confidence. Our analysis demonstrates that RNA editing is a strong discriminative tool for examining sensitivity of melanoma patients to immunotherapy.     

Isolation of the melanoma-associated antigen p23 using antibody phage display

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.     

Analysis of CRISPR‐Cas9 screens identifies genetic dependencies in melanoma

Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR‐Cas9 genetic screens provide a genome‐wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR‐Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type‐specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.     

Innovative Therapy,Monoclonal Antibodies and Beyond

The seventh Edition of “Innovative Therapy, Monoclonal Antibodies and Beyond” Meeting took place in Milan, Italy, on January 27, 2017. The two sessions of the meeting were focused on: 1) Preclinical assays and novel biotargets; and 2) monoclonal antibodies, cell therapies and targeted molecules. Between these two sessions, a lecture entitled “HLA-antigens modulation and response to immune checkpoint inhibitor immunotherapy” was also presented. Despite the impressive successes in cancer immunotherapy in recent years, the response to immune based interventions occurs only in a minority of patients (∼20%). Several basic and translational mechanisms of resistance to immune checkpoint blockers (ICBs) were discussed during the meeting: 1. the impact of tumor microenvironment on the activity of immune system; 2. strategies to inhibit the cross-talk between extracellular matrix and myeloid-derived suppressor cells (MDSC) in the preclinical setting; 3. microRNA expression as a biomarker and as a target of therapy in non-small cell lung cancer (NSCLC); 4. the significance of complement activation pathways in response to immune checkpoint inhibitors; 5. the immunosuppressive activity of the microbiota by inducing IL-17 producing cells; and 6. modulation of HLA antigens as possible markers of response to ICB therapy. In order to overcome the deficiency in active anti-tumor T cells, several clinically applicable combination strategies were also discussed: 1. strategies to enhance the anticancer effects of immunogenic cell death inducing-chemotherapy; 2. the use of CAR T-cells in solid tumors; 3. the use of combination strategies involving oncolytic viruses and ICBs; 4. combinations of new ICBs with anti-PD-1/CTLA-4 therapy; and 4. combinations of targeted therapies and ICBs in melanoma. Overall, this conference emphasized the many novel strategies that are being investigated to improve the overall patient response to cancer immunotherapy. Optimization of biomarkers to accurately select patients who will respond to immunotherapy, coupled with combination strategies to improve long term patient survival remain critical challenges in the immuno-oncology field.     

Lymphocyte-mediated cytotoxicity: effects of ageing, adult thymectomy and thymic factor.

Adult thymectomy, as well as ageing, depressed splenic lymphocyte-mediated cytotoxicity (LMC) in the mouse. Ageing depressed significantly LMC as early as 19 weeks of age, independently of the number of cells used for immunization. Thymectomy affected LMC only when supoptimal numbers of immunizing allogeneic cells were used. This effect peaked at 6 to 12 weeks after thymectomy. No difference between thymectomized and normal mice was observed when LMC was tested 16 to 20 weeks after thymectomy, at an age when normal control mice themselves already showed a lowered LMC due to ageing. The effect of in vivo treatment with a circulating thymic factor (TF), which was shown to disappear with ageing as well as after adult thymectomy, has been tested in adult thymectomized mice and normal young and ageing mice. TF treatment prevented LMC depression in adult thymectomized mice, whereas it depressed paradoxically splenic LMC in normal young and old mice. The possible mechanisms of the effects of adult thymectomy, ageing, and thymic factor on the different T cell subsets involved in allogeneic killer cell generation are discussed.     

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.     

Dendritic cell-based combined immunotherapy with autologous tumor-pulsed dendritic cell vaccine and activated T cells for cancer patients: rationale, current progress, and perspectives

Effective adoptive cancer immunotherapy depends on an ability to generate tumor-antigen-presenting cells and tumor-reactive effector lymphocytes and to deliver these effector cells to the tumor. Dendritic cells (DCs) are the most potent antigen-presenting cells, capable of sensitizing T cells to new and recall antigens. Many studies have shown that tumors express unique proteins that can be loaded on DCs to trigger an immune response. The current experimental and clinical statuses of adoptive transfer of tumor antigen-pulsed DCs and vaccine-primed activated T cells are summarized herein. Clinical trials of antigen-pulsed DCs have been conducted in patients with various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies have shown that antigen-loaded DC vaccination is safe and promising for the treatment of cancer. In addition, tumor vaccine-primed T cells have been shown to induce antitumor activity in vivo. Several clinical studies are being conducted on the use of vaccine-primed T cells such as tumor-drainage lymph node. It is reasonable to consider using both tumor antigen-pulsed DCs and vaccine-primed lymphocytes as adjuvants. We are now investigating the use of autologous whole tumor antigen-pulsed DCs and the DC vaccine-primed activated lymphocytes in patients with multiple metastasis of solid tumors.