Immune signatures in cutaneous melanoma correlate with survival independently of immunotherapy treatment

Immune checkpoint inhibitors (ICIs) have fundamentally improved survival from advanced cutaneous melanoma. Significant efforts have been made to understand the ICI response to identify ways to further improve outcomes. One such approach has been to investigate gene expression associated with response to ICI, which has identified various immune-related mRNA signatures, including a six-gene IFN-γ signature (IFN-γ⁶), an expanded immune signature (IFN-γ¹⁸), an effector T-cell gene signature (T_eff), and a T_eff-associated and IFN-γ-associated gene signature (T_eff + IFN-γ). Given that these signatures appear to reflect expression from T cells and the level of tumour-infiltrating immune cells has been associated with survival, we hypothesised that the prognostic value of the signatures is not limited to ICI treatment and investigated if they were associated with survival also in patients who never received ICI. The signatures were not present in melanoma cell lines when compared with tumour samples, confirming that the signatures were likely derived from the samples' non-tumour (immune) components. We acquired expression and survival data from five melanoma cohorts with a wide range of disease stages, treatments and metrics for survival, and correlated the expression signatures with survival. All four signatures were significantly associated (p < .05) with survival in four of five cohorts, with hazard ratios ranging from 0.69 to 0.92. We conclude that these immune signatures' association with survival is not specific to ICI-treated patients, but present in a number of settings.     

Identification immunophenotyping of lung adenocarcinomas based on the tumor microenvironment

The tumor immune microenvironment is heterogeneous, and its impact on treatment responses is not well understood. It is still a challenge to analyze the interaction between malignant cells and the tumor microenvironment to apply suitable immunotherapy in lung adenocarcinoma. We performed the nonnegative matrix factorization method to 513 messenger RNA expression profiles of lung adenocarcinomas (LUADs) from The Cancer Genome Atlas (TCGA) to obtain an immune-related expression pattern. Subsequently, we characterized the immune-related gene signatures and clinical and survival characteristics. We used 576 patients from Gene Expression Omnibus to confirm our findings. Of the patients in the training cohort, 51% had a high immune enrichment score, high expression of immune cell signaling, cytolytic activity, and interferon (IFN)-related signatures (all P < .05). We denoted these as the Immune Class. We further subdivided the Immune Class into two subclasses based on the tumor microenvironment. These were denoted the Active Immune Class and Exhausted Immune Class. The former showed significant IFN, T-cells, M1 macrophage signatures, and better prognosis (all P < .05), while the latter presented an exhausted immune response with activated stromal enrichment, M2 macrophage signatures, and immunosuppressive factors such as WNT/transforming growth factor-β (all P < .05). Furthermore, we predicted the response of our immunophenotypes to immunological checkpoint inhibitors (P < .05). Our findings provide a novel insight into the immune-related state of LUAD and can identify the patients who will be receptive to suitable immunotherapeutic treatments.     

Tumoricidal activity of syngeneic macrophages from melanoma-bearing mice: Changes with progressive tumor growth

Summary Changes in the cytostatic and cytotoxic activity of macrophages from tumor-bearing (TBM) and control mice were studied in a murine model of malignant melanoma. Syngeneic macrophages from TBM were initially noncytotoxic, but became cytotoxic and achieved their maximum destructive ability after 14 days of tumor growth. With continued tumor growth these macrophages either lost or had reduced cytotoxic activity. In contrast, macrophages from the same melanoma-bearing animals were significantly cytostatic at an earlier stage of tumor growth, but with continued melanoma growth these macrophages were no more cytostatic than controls. Moreover, melanomas grew slowly during the time when macrophages were observed to be cytostatic but grew rapidly at those stages when macrophages had a reduced ability to inhibit melanoma DNA synthesis. When these effector cells became cytotoxic melanomas were growing rapidly and changes in cytotoxicity had little effect on tumor mass. Thus, macrophages do not completely suppress melanoma proliferation and, although exhibiting cytotoxicity they were relatively ineffective in controlling a large mass of tumor cells.     

Probing immune infiltration dynamics in cancer by in vivo imaging

Cancer immunotherapies typically aim to stimulate the accumulation and activity of cytotoxic T-cells or pro-inflammatory antigen-presenting cells, reduce immunosuppressive myeloid cells or regulatory T-cells, or elicit some combination of effects thereof. Notwithstanding the encouraging results, immunotherapies such as PD-1/PD-L1-targeted immune checkpoint blockade act heterogeneously across individual patients. It remains challenging to predict and monitor individual responses, especially across multiple sites of metastasis or sites of potential toxicity. To address this need, in vivo imaging of both adaptive and innate immune cell populations has emerged as a tool to quantify spatial leukocyte accumulation in tumors non-invasively. Here we review recent progress in the translational development of probes for in vivo leukocyte imaging, focusing on complementary perspectives provided by imaging of T-cells, phagocytic macrophages, and their responses to therapy.     

Fine needle aspirate smear morphology in metastatic melanoma

Cytologic smear preparations of fine needle aspirates obtained from 71 patients with biopsy-proven metastatic malignant melanoma were morphologically analyzed. Cell diameter, percentage of cells lying individually, smear cellularity, mitotic rate, percentage of cells with nuclear inclusions and number of giant cells were quantitated. Qualitative estimates of degree of pigmentation, background composition, presence of macrophages, presence of contaminating blood, cell shape, amount and appearance of cytoplasm, prominence of Golgi apparatus, reactivity with the antibody NKI/C-3 and eccentricity of the nuclei were made. While eccentric nuclei, cytoplasmic vacuoles, intranuclear inclusions, high cellularity and cells with abundant cytoplasm were found frequently in smears from malignant melanomas, the presence of reactivity with NKI/C-3, pigment, giant cells, a bloody background and a lack of cell cohesion were the features most commonly associated with smears obtained from patients harboring metastatic melanoma.     

Antimelanoma Antibodies in Swine With Spontaneously Regressing Melanoma

Sinclair swine provide a unique model for studying mechanisms of tumor regression because they are born with melanomas that spontaneously regress approximately 10 weeks after birth. To examine whether an antitumor immune response is present in these animals, and, if so, to study its relation to tumor regression, 38 sera specimens collected at different times from 13 swine born with melanomas were tested for melanoma antibodies by immunoprecipitation and SDS-PAGE analysis of ¹²⁵I labelled swine melanoma macromolecules. Antibodies to melanoma were present in 13 (100%) of the swine versus 1 of 3 control swine. The antibodies were directed to antigens of approximately 45, 68–75, or 100 kDa. These antigens were also expressed on human melanomas and normal melanocytes but on only one of five unrelated tumors. The incidence and level of these antibodies increased with time. Antibodies to the 45, 68–75, and 100 kDa antigens were present in 36%, 55%, and 9%, respectively, of sera collected prior to 7 weeks of age, but in 80%, 100%, and 37% of sera collected between 7 and 20 weeks (P<0.05). The rise in melanoma antibodies usually preceded or appeared together with tumor regression and loss of pigmentation. These findings indicate that Sinclair swine with melanomas have antibodies to antigens preferentially expressed on pigment cells, and support the hypothesis that the regression phenomenon and the vitiligo-like skin depigmentation result from immune responses to common antigens shared by normal and malignant swine pigment cells.     

Expression of MAGE-C1/CT7 and MAGE-C2/CT10 predicts lymph node metastasis in melanoma patients

MAGE-C1/CT7 and MAGE-C2/CT10 are members of the large MAGE family of cancer-testis (CT) antigens. CT antigens are promising targets for immunotherapy in cancer because their expression is restricted to cancer and germ line cells and a proportion of cancer patients presents with immune responses against CT antigens, which clearly demonstrates their immunogenicity. This study investigates the expression of MAGE-C1/CT7 and MAGE-C2/CT10 in primary and metastatic melanoma. Immunohistochemical staining of tissue microarrays that consisted of 59 primary malignant melanomas of the skin, 163 lymph node and distant melanoma metastases and 68 melanoma cell lines was performed. We found MAGE-C1/CT7 expression in 15 out of 50 (24%) primary melanomas and 15 out of 50 (24%) cell lines, whereas MAGE-C2/CT10 was detected in 17 out of 51 (33%) primary melanomas and 14 out of 68 (17%) cell lines. MAGE-C1/CT7 and MAGE-C2/CT10 were both detected in 40% of melanoma metastases. Patients with MAGE-C1/CT7 or MAGE-C2/CT10 positive primary melanoma had significantly more lymph node metastases (p = 0.005 and p<0.001, resp.). Prediction of lymph node metastasis by MAGE-C1/CT7 and MAGE-C2/CT10 was independent of tumor cell proliferation rate (Ki67 labeling index) in a multivariate analysis (p = 0.01). Our results suggest that the expression of MAGE-C1/CT7 and MAGE-C2/CT10 in primary melanoma is a potent predictor of sentinel lymph node metastasis.     

Induction of immune response against NY-ESO-1 by CHP-NY-ESO-1 vaccination and immune regulation in a melanoma patient

BACKGROUND: NY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro. AIM: In this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites. RESULTS: Ten days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-gamma secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4(+)CD25(+)Foxp3(+) Tregs was observed in PBMC but significantly increased numbers of CD4(+)CD25(+)Foxp3(+) Tregs and CD68(+) immunoregulatory macrophages were detected at the local tumor sites. CD4(+)CD25(+)Foxp3(+) Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site. CONCLUSIONS: Our observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.     

Galectin-1-mediated biochemical controls of melanoma and glioma aggressive behavior

Gliomas and melanomas are associated with dismal prognosis because of their marked intrinsic resistance to proapoptotic stimuli,such as conventional chemotherapy and radiotherapy,as well as their ability to escape immune cell attacks.In addition,gliomas and melanomas display pronounced neoangiogenesis.Galectin-1 is a hypoxia-sensitive protein,which is abundantly secreted by glioma and melanoma cells,which displays marked proangiogenic effects.It also provides immune tolerogenic environments to melanoma and glioma cells through the killing of activated T cells that attack these tumor cells.Galectin-1 protects glioma and melanoma cells against cytotoxic insults(including chemotherapy and radiotherapy) through a direct role in the unfolded protein response.Altogether,these facts clearly point to galectin-1 as an important target to be combated in gliomas and melanomas in order to:(1) weaken the defenses of these two types of cancers against radiotherapy,chemotherapy and immunotherapy/vaccine therapy;and(2) reinforce antiangiogenic therapies.In the present article,we review the biochemical and molecular biology-related pathways controlled by galectin-1,which are actually beneficial for melanoma and glioma cells,and therefore detrimental for melanoma and glioma patients.     

Comparative Histopathology of Grey‐Horse‐Melanoma and Human Malignant Melanoma

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S‐100, proliferating cell nuclear antigen (PCNA), HMB‐45, Ki‐67, T‐311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.     

Comparative histopathology of grey-horse-melanoma and human malignant melanoma

Equine melanoma shows striking features particularly with regard to clinical development in grey horses: in contrast to malignant melanoma in humans and in solid coloured horses that are characterized by early onset of metastasis, pigment cell tumours display almost benign clinical features in ageing grey horses. Through evolution, grey horses appear to be in a favourable position in regard to the biological behaviour of melanomas. Yet unknown factors inhibiting or retarding early melanoma metastasis may be responsible for this phenomenon. In this study, immunostaining profiles and histopathologic patterns of equine vs. human melanotic tumours were compared. In addition, the expression of melanoma markers currently used in human melanoma detection and characterization were evaluated for their applicability in equine melanoma diagnosis. Immunohistopathologic investigations revealed that benign grey horse melanomas share common features with human blue nevi and with human malignant desmoplastic melanomas, whereas their resemblance to other types of human cutaneous malignant melanomas is less pronounced. Our data equally underline that S-100, proliferating cell nuclear antigen (PCNA), HMB-45, Ki-67, T-311 and CD44 can serve as reliable markers for horse melanomas. Further investigations aiming at identifying factors retarding metastasis in affected grey horses are needed, as they may contribute to the development of novel treatment strategies for human malignant melanoma.     

Assessment of clinical studies evaluating combinations of immune checkpoint inhibitors with locoregional treatments in solid tumors

In the last decade, immunotherapy with immune checkpoint inhibitors (ICIs) has changed the therapeutic algorithm of cancer patients. ICIs combined with other therapeutic options, such as chemo- and targeted therapies, generate impressive results in cancer patients. Locoregional treatments (LRTs) play an important role in the management of various solid tumors (e.g., hepatocellular carcinoma (HCC), neuroendocrine tumors, etc.), and this therapeutic approach may enhance the activity of the immune response to tumor cells destroying primary tumors and leading to the release of several soluble molecules. This systematic review was performed to identify studies reporting objective response rate (ORR) and survival information in patients with solid tumors treated with ICIs plus LRTs. In the present work, fourteen studies were included, and the majority of them (five studies) enrolled patients with hepatocellular carcinoma (HCC), whereas the others included patients with different diseases. The highest ORRs were seen in HCC (67%, Y-90 RE plus ipilimumab and nivolumab) and melanoma (38%, dendritic cells with mRNA plus ipilimumab) patients. ORRs were not observed in liver metastases from melanoma and colorectal cancer. These data suggest that combination of ICIs and LRTs is feasible and more active in primary tumors (particularly HCC) than metastases with a synergistic effect on antitumor immunity. However, further studies are needed to better select patients, schedules, and setting of treatments.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole‐exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer–testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell‐based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.     

PLX3397 inhibits the accumulation of intra‐tumoral macrophages and improves bromodomain and extra‐terminal inhibitor efficacy in melanoma

Bromodomain and extra‐terminal inhibitors (BETi) delay tumor growth, in part, through tumor cell intrinsic alterations and initiation of anti‐tumor CD8+ T‐cell responses. By contrast, BETi effects on pro‐tumoral immune responses remain unclear. Here, we show that the next‐generation BETi, PLX51107, delayed tumor growth to differing degrees in Braf V600E melanoma syngeneic mouse models. These differential responses were associated with the influx of tumor‐associated macrophages during BETi treatment. Tumors that were poorly responsive to PLX51107 showed increased influx of colony‐stimulating factor‐1 receptor (CSF‐1R)‐positive tumor‐associated macrophages. We depleted CSF‐1R+ tumor‐associated macrophages with the CSF‐1R inhibitor, PLX3397, in combination with PLX51107. Treatment with PLX3397 enhanced the efficacy of PLX51107 in poorly responsive Braf V600E syngeneic melanomas in vivo. These findings suggest that tumor‐associated macrophage accumulation limits BETi efficacy and that co‐treatment with PLX3397 can improve response to PLX51107, offering a potential novel combination therapy for metastatic melanoma patients.     

Peripheral immune cell gene expression predicts survival of patients with non-small cell lung cancer

Prediction of cancer recurrence in patients with non-small cell lung cancer (NSCLC) currently relies on the assessment of clinical characteristics including age, tumor stage, and smoking history. A better prediction of early stage cancer patients with poorer survival and late stage patients with better survival is needed to design patient-tailored treatment protocols. We analyzed gene expression in RNA from peripheral blood mononuclear cells (PBMC) of NSCLC patients to identify signatures predictive of overall patient survival. We find that PBMC gene expression patterns from NSCLC patients, like patterns from tumors, have information predictive of patient outcomes. We identify and validate a 26 gene prognostic panel that is independent of clinical stage. Many additional prognostic genes are specific to myeloid cells and are more highly expressed in patients with shorter survival. We also observe that significant numbers of prognostic genes change expression levels in PBMC collected after tumor resection. These post-surgery gene expression profiles may provide a means to re-evaluate prognosis over time. These studies further suggest that patient outcomes are not solely determined by tumor gene expression profiles but can also be influenced by the immune response as reflected in peripheral immune cells.     

Decreased expression of Apaf-1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease-activating factor-1 (Apaf-1) is a cell death effector that acts with cytochrome c and caspase-9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf-1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf-1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf-1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf-1 expression was observed when comparing nevi and melanomas (chi-square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf-1 (chi-square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf-1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf-1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf-1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.