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DNA hypomethylating agents (DHA) combined with immune-checkpoint inhibitors (ICI) is a promising strategy to improve the effect of immunotherapy. Providing initial support to this notion, the phase Ib NIBIT-M4 study, a dose-escalation trial of the DHA guadecitabine plus ipilimumab, has shown this regimen to be safe, tolerable and with promising immunomodulatory and anti-tumor activity in advanced melanoma. Here we report the 5-year clinical outcome of NIBIT-M4 patients and its correlation with an integrated multi-omic analysis. The NIBIT-M4 enrolled unresectable Stage III or IV cutaneous melanoma patients. Median Overall Survival (OS), Progression Free Survival (PFS), 5-year OS and PFS rate, and median Duration of Response (DoR) were assessed. Tumor biopsies were performed at baseline and at week 4 and 12 on-treatment. RNA-seq, whole exome sequencing (WES), RRBS methylation profiling, and tumor immune contexture data generated by immunohistochemistry (IHC), were integratively analyzed and correlated with clinical outcome. Patients were classified as responder (R) or non-responder (NR) based on Disease Control. Nineteen patients were enrolled in the NIBIT-M4 study between October 2015, and August 2018. As of July 1, 2022, median OS was 25.6 months (mo) (95% CI, 0.0-52.9), median PFS was 5.2 mo (95% CI, 4.0-6.4); 5-year OS rate was 28.9% and 5-year PFS rate was 5.3 %; median DoR was 20.6 mo (95% CI, 12.4-28.8). Tumor biopsies for correlative analyses were available from 14 patients. Compared to NR, R patients showed higher mutation frequency in NRAS, while NR patients harbored more frequent somatic alterations in genes involved in Epithelial to Mesenchymal Transition (EMT) and chromatin organization pathways. By tumor transcriptome analysis, NR patients showed enrichment for proliferation/differentiation/EMT pathways associated with suppression of INF-ɣ/HLA-II/immune-related transcriptional modules. R patients displayed a dynamic pattern of activated immune signatures related to CD8+ Tex cells, B cells, tertiary lymphoid structures, TFH cells and IFN-ɣ, reaching highest expression levels in week 12 biopsies. Deconvolution of transcriptomic data showed higher CD8+ and NK cell content and higher correlation of TCRB clonality with CD8+ content in R patients. By integration of a genetic immunoediting index (GIE) with an adaptive immunity signature (ICR) lesions were stratified into four distinct subsets. Interestingly, the ICR/GIE classification discriminated 5-year OS and PFS while the classification based on response groups did not. Moreover, patients with a “high ICR/non-GIE” (i.e., without immunoediting) showed lower expression of antigen presentation and processing-related genes associated with defective HLA-class I expression in the lesions, in spite of high CD8+ content. Citation Format: Teresa Noviello, Anna Maria Di Giacomo, Francesca Pia Caruso, Alessia Covre, Giovanni Scala, Luigi Ferraro, Sandra Coral, Wolf H. Fridman, Catherine Sautes-Fridman, Roberta Mortarini, Elena Simonetti, Maria Fortunata Lofiego, Davide Bedognetti, Andrea Anichini, Michele Ceccarelli, Michele Maio. Guadecitabine plus ipilimumab in unresectable melanoma: Five-year follow-up and correlation with integrated, multi-omic analysis in the NIBIT-M4 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3273.

The immune response to melanoma improves survival in untreated patients and predicts response to immune checkpoint blockade. Here we report genetic and environmental predictors of the immune response in a large primary cutaneous melanoma cohort. Bioinformatic analysis of 703 tumor transcriptomes was used to infer immune cell infiltration and categorize tumors into immune subgroups, which were then investigated for association with biological pathways, clinicopathological factors, and copy number alterations. Three subgroups, with "Low”, “Intermediate", and "High" immune signals were identified in primary and replicated in metastatic tumors. Genes in the Low subgroup were enriched for cell cycle and metabolic pathways, whereas genes in the High subgroup were enriched for interferon and NF-κB signaling. We identified high MYC expression partially driven by amplification, HLA-B downregulation, and deletion of IFN-γ and NF-κB pathway genes as regulators of immune suppression. Furthermore, we showed that cigarette smoking, a globally detrimental environmental factor, modulates immunity, reducing survival primarily in patients with a strong immune response. Together these analyses identify a set of easily assessible factors that may serve as predictors of response to immunotherapy in melanoma patients.

Background: Therapeutic targeting of the PD-1/PDL-1 axis significantly increases the survival of melanoma (MM) and non-small cell lung cancer (NSCLC) patients. Despite this unprecedented efficacy, a sizeable proportion of MM and NSCLC patients fails to benefit from therapy due to primary or secondary resistance to treatment. In this scenario, deepening the knowledge on mechanism(s) underlying treatment failure allows to design novel, mechanism-based, therapeutic approaches to overcome resistance to anti-PD-1/PDL-1 therapy. Along this line, we have extensively characterized the immunomodulatory properties of DNA hypomethylating agents (DHA) in different human malignances. Exposure of neoplastic cells to DHA efficiently improved antigen-restricted and -unrestricted T cell recognition of cancer cells in vitro through the upregulation/induction of the expression of epigenetically regulated tumor associated antigens, HLA class I and/or accessory/co-stimulatory molecules by neoplastic cells. Supporting these ex vivo data, combining the systemic administration of DHA (ie, decitabine or guadecitabine) with antibodies to different immune checkpoints significantly reduced tumor growth of murine syngeneic grafts, compared to single-agent therapy. Supporting these pre-clinical findings, our phase Ib NIBIT-M4 study has been the first clinical trial demonstrating that systemic administration of guadecitabine followed by CTLA-4 blockade with ipilimumab is safe and tolerable in MM patients, shows a promising anti-tumor, and induces a significant modulation of immune related pathways in tumor samples (Di Giacomo AM, Clin Cancer Res 2019). Prompted by these results and to further explore the efficacy of DHA combined with immune-checkpoints blockade we have designed and activated the NIBIT-ML1 trial. This study aims at investigating the efficacy of guadecitabine combined with ipilimumab and nivolumab in reverting the resistance to PD-1/PDL-1 therapy of MM and NSCLC patients. Methods: The NIBIT-ML1 is a randomized, phase II study designed according to the two stages optimal design by Simon, in unresectable Stage III or Stage IV MM (Cohort A) or NSCLC (Cohort B) patients who failed therapy with anti-PD-1/PDL-1 as last treatment. Primary objective of the study is immune (i)-ORR according to iRECIST criteria. Secondary objectives include safety, DCR, PFS, median OS, and survival rate at 1 and 2-years. Extensive cellular and molecular immune correlates will also be explored. Following a safety run-in phase in 6 subjects per Cohort, eligible patients will be randomized to receive: guadecitabine plus ipilimumab and nivolumab (ARM A) or ipilimumab and nivolumab (ARM B). Sample size will range from 6 to 92 patients per Cohort. The first patient first visit is foreseen by March 2020 (NCT04250246) Citation Format: Anna Maria Di Giacomo, Luana Calabrò, Riccardo Danielli, Monica Valente, Elisabetta Gambale, Sandra Coral, Giovanni Amato, Harold Keer, Diana Giannarelli, Mohammad Azab, Andrea Anichini, Alessia Covre, Michele Maio. A randomized, multi-center, phase II study of nivolumab combined with ipilimumab and guadecitabine or nivolumab combined with ipilimumab in melanoma and NSCLC patients resistant to anti-PD-1/-PD-L1: The NIBIT-ML1 Study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT270.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed “early effector cells” (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non–small cell lung cancer. Cancer Res; 77(4); 851–61. ©2016 AACR.

CD8+ T cells at the earliest stage of effector generation have not been identified at tumor site of melanoma patients. Such early effectors, if present, should be characterized by a specific phenotype, distinct from that expressed at later stages of the antigen-induced differentiation program, by short-lived effector cells, memory precursors, and terminal effectors. Here, we show that neoplastic tissues from primary and metastatic lesions of melanoma patients contain a subset of CD8+ T cells expressing FOXP3. CD8+ FOXP3+ CD25+ T lymphocytes were found in tumor-invaded lymph nodes (TILN), s.c. metastases, and advanced primary lesions. Their frequency was significantly higher in TILN compared with tumor-free lymph nodes or with peripheral blood and in primary tumors compared with TILN. CD8+ FOXP3+ T cells did not express markers of regulatory [CTLA-4, CCL4, interleukin-10 (IL-10), transforming growth factor-β1], exhausted (PD-1), or senescent (CD57) CD8+ T lymphocytes. Instead, this subset showed an antigen-experienced “EM1” phenotype (CCR7− CD45RA− CD28+ CD27+) and exhibited a CD127−, KLRG1−, HLA-DR+, CD38+, T-bet+, perforin+ “early effector” profile predicted by current models. CD8+ FOXP3+ T cells produced IFN-γ on short in vitro activation, recognized autologous tumor by CD107a mobilization, and expressed Ki-67 on ex vivo analysis. In response to autologous tumor plus IL-2/IL-15, the CD8+ FOXP3+ T cells proliferated promptly and showed competence for differentiation (downregulation of CD27 and upregulation of T-bet). These results suggest development of early phases of antitumor immunity even in advanced melanoma. Moreover, the CD8+ FOXP3+ “early effector” subset may be an invaluable tool for monitoring immunity at tumor site. Cancer Res; 70(21); 8378–87. ©2010 AACR.

Introduction Tumor microenvironment plays an essential role in cancer development and progression. In the context of thyroid cancer (TC) it still represents a poorly investigated field. Recent data from in vitro and in vivo studies proposed a model of TC progression promoted by the converging action of thyroid tumor cells and cancer-associated fibroblasts (CAFs). According to this mouse model CAFs are recruited in tumor stroma where synthesize and deposit collagen (COL1A1) that is, in turn, cross-linked by the enzyme LOX, produced by the thyroid tumor cells. Collectively this coordinated action lead to matrix stiffness and TC progression. Interestingly, this process is reported to be specifically observed in murine thyroid tumors expressing BRAFV600E mutation, but not in those harboring RAS mutation. In this study we tested this cross-talk in human thyroid cancer tissues. Experimental procedures A series of thyroid specimens, comprising different tumor subtypes (PTC and PDTC) and histologic variants, was collected at our institution and investigated by immunohistochemical (IHC) analyses for the expression of α-SMA (marker for activated fibroblasts), collagen COL1A1 and LOX. Tumor samples were also screened for the most common genetic lesions reported in TC including BRAFV600E and N/H/KRAS mutations. Publicly available datasets of thyroid tumors and non-neoplastic thyroid controls, analyzed on Affymetrix HG-U133 Plus 2.0 array, were selected to investigate the expression profiles of α-SMA, COL1A1 and LOX genes. Robust Multi Array Average (RMA) normalization, Brain Array annotation and batch correction procedures were applied to obtain gene expression profiles across all samples. Results In IHC analyses α-SMA decoration was observed in the stroma of thyroid tumor tissues while was restricted to blood vessels in the adjacent non-neoplastic thyroid. Stromal α-SMA staining was not evenly distributed in the tumors but localized preferentially at the tumor invasive border, organized in peripheral structures as fibrous septa or capsule. In agreement with the proposed model, higher level of α-SMA staining were specifically observed in PTC tumors harboring BRAFV600E mutation compared with tumors harboring RAS mutation. IHC analyses on tissue serial sections confirmed the coordinated expression of α-SMA, COL1A1 and LOX; COL1A1 localized in tumor stroma and was closely correlated with α-SMA positive areas while LOX was expressed by thyroid tumor cells. The coordinated expression of the three genes was confirmed by meta-analysis of public datasets. Conclusions In this study we provide evidence of the coordinated presence of thyroid tumor cells, activated fibroblasts and proteins of extracellular matrix, such as COL1A1 and LOX, in the contest of human thyroid carcinoma. Citation Format: Emanuela Minna, Silvia Brich, Katia Todoerti, Silvana Pilotti, Federica Perrone, Nicholas Paielli, Luca Agnelli, Ilaria Bersani, Roberta Mortarini, Andrea Anichini, Paola Collini, Antonino Neri, Angela Greco, Maria Grazia Borrello. Investigation of thyroid tumor microenvironment by immunohistochemical and bioinformatic analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4071.

Activating mutations in BRAF and NRAS oncogenes in human melanomas are mutually exclusive. This finding has suggested an epistatic relationship but is consistent even with synthetic lethality. To evaluate the latter possibility, a mutated NRASQ61R oncogene was expressed, under a constitutive or a doxycycline-regulated promoter, in a metastatic melanoma clone (clone 21) harboring an activated BRAFV600E oncogene. After the first 10 to 12 in vitro passages, the constitutive NRASQ61R transfectant displayed progressive accumulation in G0-G1 phase of the cell cycle and stained for the senescence-associated β-galactosidase activity (SA-β-Gal). Inducible expression of NRASQ61R, by the Tet-Off system, in clone 21 cells (21NRAS61ON) led to overactivation of the RAS/RAF/mitogen-activated protein kinase signaling pathway and, after the 10th in vitro passage, led to promotion of senescence. This was documented by reduced proliferation, flattened cell morphology, reduced growth in Matrigel, positive staining for SA-β-Gal, and expression of AMP-activated protein kinase and of the cell cycle inhibitor p21waf1/Cip1. These effects were detected neither in 21 cells with silenced NRASQ61R (21NRAS61OFF) nor in cells transfected with an inducible wild-type NRAS gene (21NRASWTON). In addition, when compared with parental 21 cells, or with 21NRAS61OFF, 21NRAS61ON and constitutive NRASQ61R transfectants cells showed increased susceptibility to cytotoxicity by both HLA class I antigen–restricted and nonspecific T cells and up-regulation of several MHC class I antigen processing machinery components. These results suggest a relationship of synthetic lethality between NRAS and BRAF oncogenes, leading to selection against “double-mutant” cells. (Cancer Res 2006; 66(13): 6503-11)

Because changes in the expression level of antigen-processing machinery (APM) components and HLA class I and II antigens in melanoma cells are expected to affect their interactions with the immune system of the host, we assessed the clinical relevance of quantitative variations in the expression of these molecules in melanoma lesions. Short-term (

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALKhigh 12.8%, ALKlow 73%, P = 0.0035; cell death: ALKhigh 56.4%, ALKlow 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs. [Cancer Res 2009;69(18):7338–46]

Doxorubicin treatment was found to augment the expression of the extracellular matrix (ECM) protein fibulin-1 in cultured human breast cancer cell lines and in MDA-MB-361 tumors grown in athymic mice. Doxorubicin was also found to augment tumor expression of the fibulin-1–binding proteins fibronectin and laminin-1. Growth of breast cancer cell lines on Matrigel, an ECM extract containing fibulin-1 and laminin-1, resulted in lower levels of doxorubicin-induced apoptosis as compared with controls. Moreover, tumors formed by injection of athymic mice with MDA-MB-361 cells mixed with Matrigel were significantly more doxorubicin resistant and displayed lower levels of apoptosis compared with those that formed in the absence of Matrigel. Monoclonal antibodies against fibulin-1 reversed Matrigel-dependent doxorubicin resistance. Furthermore, small interfering RNA–mediated suppression of fibulin-1 expression in breast cancer cells resulted in a 10-fold increase in doxorubicin sensitivity as compared with control cells. Together, these findings point to a role for fibulin-1 in breast cancer chemoresistance. [Cancer Res 2007;67(9):4271–7]

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1–dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1− cells) nor up-regulated (in APAF-1+ cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1+ and APAF-1− tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 –specific inhibitors in both APAF-1+ and APAF-1− tumor cells. In response to 1 to 100 μmol/L of cisplatin, camptothecin, or celecoxib, APAF-1+ melanomas (n = 12) did not show significantly increased levels of apoptosis compared with APAF-1− tumors (n = 7), with the exception of enhanced apoptosis in response to a very high dose (100 μmol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.

Activation of CTL-mediated antitumor immunity to self-epitopes expressed by neoplastic cells is thought to be prevented, at any stage of tumor progression, by tolerance mechanisms. In contrast, in 74 American Joint Committee on Cancer stages I-IV melanoma patients, we found that development of lymph node metastases is a key event triggering CD8(+) T-cell-mediated immunity to self-epitopes encoded by melanocyte differentiation antigens. This was shown by the increased peripheral precursor frequency to Melan-A/Mart-1, gp100, and tyrosinase epitopes in stage III and IV compared with stage I and II patients, and by accumulation of functional memory T cells directed to Melan-A/Mart-1(26-35) in tumor-invaded lymph nodes. However, in tumor-invaded lymph nodes of most patients, CD8(+) T cells directed to melanocyte differentiation antigens or to tumor-restricted antigens (MAGE-3 and NY-ESO-1 epitopes), showed a CCR7(+) CD45RA(+) CD27(+) CD28(+) perforin(-) "precursor" phenotype. Only in 7 of 23 cases antigen-specific CD8(+) T cells in invaded lymph nodes showed a predominant CCR7(-) CD45RA(-) CD27(+) CD28(-) perforin(+) "preterminally differentiated" phenotype. In the latter subset of patients, by immunohistochemistry in lymph node lesions, we found that CD8(+) T lymphocytes intermingling with the neoplastic tissue expressed a CCR7(-) CD45RO(+)/RA(-) phenotype, whereas CD4(+) lymphocytes did not infiltrate the tumor. Furthermore, perforin and granzyme B were expressed on a higher fraction of the CD8(+) cells surrounding the invading tumor compared with the lymphocytes infiltrating the neoplastic tissue. In addition, no evidence for tumor regression was found in such metastatic lesions, as documented by absence of neoplastic cell necrosis or apoptosis. These data indicate that neoplastic cells in the lymph nodes and/or increased tumor burden in metastatic disease activate CD8(+) T-cell-mediated antitumor immunity to self-epitopes. However, the paucity of terminally differentiated CD8(+) T cells at tumor site suggests that immunotherapy strategies may require not only the boosting of tumor immunity, but also effective means to promote CD8(+) T-cell differentiation in the neoplastic tissue.

Purpose: Chemotherapy (CT) with Dacarbazine (DTIC) in association with an anti-VEGF treatment could be an intriguing exploiting path, since metastatic melanoma (MM) in vitro models showed VEGF as a possible target, due to its transcription up-regulation by DTIC. The purpose of this study was to indentify modulated circulating factors which may be used as specific biomarkers in patients (pts) affected by MM undergoing DTIC plus Bevacizumab (B = Avastin) combination and to test its tolerability and efficacy. Patients and Methods: Between July 2006-September 2009, 40 untreated MM pts underwent CT with DTIC (800mg/mq q4w) + B (10mg/kg q2w). Clinical benefit, response duration, safety and feasibility were analyzed. Moreover, 13 pts participating to the clinical study agreed to provide blood for translational research. The blood samples for this study were drawn at the same time points as for regular follow-up tests and were evaluated for the following several soluble factors: vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-R1, VEGF-R2, angiopoietin (Ang)-2, E-cadherin, E-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Correlations with pts clinical outcome were performed as well. Results: 36/40 pts were evaluated at the analysis-time. Median age was 57yr (male 60yr; female 47yr) and 23 were male. Three pts (8.3%) experienced a complete remission (CR), 3 pts (8.3%) developed partial response (PR), 13 (36.1%) stable disease (SD); 17 (47.2%) progressive disease (PD). Clinical benefit (CR+PR+SD) was 52.7% and treatment was well tolerated. Time to progression (months) was 10.9, respectively 22.6 for PR pts, 8.2 for SD pts and 2 for PD pts. Median-survival in the whole group was 16.5 months (PR pts 22.6; SD pts 17; PD pts 4.23). DTIC/B treatment modulates not only VEGF-A, but also VEGF-C, Ang-2 VEGF-R1 and VEGF-R2. Among soluble adhesion molecules, we observed an increase of VCAM-1 in non responder pts. Conclusions: Long-lasting responses obtained in both SD and CR/PR pts and mild side effects make DTIC/B a promising combination in advanced melanoma. The companion translational study partially clarified these clinical results by molecularly characterizing and profiling responders vs non-responders pts. Citation Format: Pier Francesco Ferrucci, Chiara Martinoli, Emilia Cocorocchio, Andrea Anichini, Sara Gandini. Translational study on circulating markers in advanced melanoma patients undergoing Dacarbazine and Bevacizumab treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2550. doi:10.1158/1538-7445.AM2014-2550

In this study, we investigated whether maturation of monocyte-derived myeloid dendritic cells (DCs) is differentially affected by the uptake of dying human melanoma cells in distinct phases of apoptosis. Maturation of monocyte-derived DCs, as documented by phenotype analysis and T-cell immunostimulatory activity, was inhibited by phagocytosis of dying melanoma cells containing a large fraction of cells in early apoptosis (Annexin-V+ and propidium iodide-) but promoted by the same tumors when in late apoptosis/secondary necrosis (Annexin-V+ and propidium iodide+) or when dying by primary necrosis. These opposite effects on DC maturation were observed after the uptake of early or late apoptotic cells from most vertical growth phase primary tumors and all metastases but not after the uptake of dying cells from a radial growth phase primary tumor or normal adult melanocytes. Inhibition of DC maturation by early apoptotic melanoma cells correlated with expression of interleukin-10 in neoplastic cells and was prevented by preincubating the tumor cells with a neutralizing antibody to interleukin-10 before tumor uptake by DCs. Cross-presentation of the melanoma-associated antigen gp100(209-217) to peptide-specific CTLs by HLA-A*0201+ DCs was achieved 48-72 h after phagocytosis of HLA-A*0201- melanoma cells in apoptosis, or primary necrosis, but only when tumor necrosis factor-alpha was added to DCs 4 h after the initiation of tumor phagocytosis. These results suggest that phases of apoptosis and neoplastic transformation affect maturation of myeloid DCs that take up dying cells of the melanocyte lineage. However, neoplastic cells in late apoptosis, or even in primary necrosis, induce only a partial DC differentiation not sufficient to achieve cross-presentation of tumor antigens to CTLs unless further DC maturation is promoted by additional signals. These results suggest a novel mechanism of tumor escape that may prevent the development of antitumor immunity through the maturation block induced in DCs by neoplastic cells in the early phase of apoptosis.

The role of endogenous NO on cell survival was investigated in human melanoma cells and melanocytes. Inducible NO synthase (iNOS) was always expressed in a panel of melanoma cell lines from metastatic lesions and in normal adult melanocytes. iNOS was also detected by immunohistochemistry in melanoma cells from metastases. Release of NO by tumor cells and melanocytes was inhibited by a specific iNOS inhibitor, aminoguanidine (AMG). Inhibition of endogenous NO synthesis did not affect cell cycle progression of melanoma cells but led to cell death by apoptosis, as indicated by Annexin V/propidium iodide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays. By contrast, iNOS inhibition by AMG did not promote apoptosis in normal adult melanocytes. A mitochondrial pathway was involved in melanoma apop tosis, as indicated by altered mitochondrial membrane potential (delta psi(m)) and down-regulation of Bcl-2 protein level after iNOS inhibition. AMG treatment triggered release of caspase-1, enzymatic activation of caspase-3, and degradation of poly(ADP-ribose) polymerase, one of the main caspase-3 substrates. Melanoma cell apoptosis induced by iNOS inhibition was completely blocked by peptide inhibitors of caspase-1 and caspase-3 (Ac-DEVD-CHO and AC-YVAD-CHO) or by an exogenous NO donor, sodium nitroprusside, or by addition of serum. Finally, comparison of control and AMG-treated melanoma cells by pathway-specific gene array analysis indicated that inhibition of NO synthesis led, before induction of apoptosis, to up-regulation of mRNA levels of genes involved in the apoptosis pathway such as Bax, caspase-1, caspase-3, caspase-6, gadd45beta, mdm2, and TRAIL. Taken together, these results indicate that melanoma cell survival is regulated by endogenous NO resulting from iNOS activity.

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-35)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.

We have shown previously that peripheral blood lymphocytes (PBL) of patients with metastatic melanoma include cytotoxic T-cell clones that recognize Melan-A/MART-1 in a HLA-A2-restricted fashion. Such clones preferentially use the variable (V) regions TCRBV14 or TCRBV7 in the beta-chain of their T-cell receptor (TCRB). It was not known, however, whether this finding is associated with the presence of the HLA-A2 allele in tumor tissue and whether evidence of the predominance of these TCRBV families can also be observed in primary tumor tissue. To address these issues, we have used a semiquantitative PCR to examine the TCRBV repertoire in six HLA-A2-matched primary melanomas in comparison with their autologous PBL. Although each patient had his or her own pattern of skewed TCRBV utilization, in all patients, T-cells that used TCRBV14 were significantly overrepresented in the neoplastic site compared with PBL. All of the primary tumors studied had detectable expression of Melan-A/MART-1 and gp100, and immunohistochemical analysis confirmed the presence of the HLA-A2 allele. Additional samples of Melan-A/MART-1-positive, gp100-positive primary melanomas from six non-HLA-A2 patients and four autologous normal skin controls failed to reveal a TCRBV14 predominance in such tissues. These results point to a role of TCRBV14 T lymphocytes in the HLA-A2-restricted immune recognition of primary melanomas.

The receptors for urokinase plasminogen activator were studied in both normal human fibroblasts (WI-38 cells) and their SV40-transformed counterpart (VA-13 cells). We have shown that transformed cells expose 10 times more urokinase plasminogen activator receptors (u-PAR) than normal cells. By cross-linking aliquots of cell lysates with the aminoterminal fragment of the A chain of u-PA, containing the receptor-binding sequence, we have observed a u-PAR concentration at focal contacts in both cell lines. Only transformed cells were able to efficiently invade the basement membrane Matrigel. Switching off the receptor gene expression by the anti-messenger oligodeoxynucleotides strategy abolished the invasive properties of transformed cells. The anti-messenger oligodeoxynucleotide sequence we have designed inhibited the u-PAR gene expression, lowering both the receptor and the receptor mRNA. This indicates that overexpression of u-PAR gene is itself responsible for invasivity of transformed fibroblasts in our cell model system and that antisense compound therapy may prove to be of clinical interest in the control of cancer spreading.

Focal adhesion kinase (FAK) is a tyrosine kinase localized at the site of focal adhesions (FA). FAK is identified as a key mediator of signaling by integrins, the major family of cell surface receptors for extracellular matrix, as well as by other receptors in both normal and cancer cells. FAK plays a prominent role in tumor progression and metastasis regulating cellular processes including migration, invasion, epithelial to mesenchymal transition, and angiogenesis. Integrin-FAK signaling has been shown to activate a number of biological mechanisms through phosphorylation and protein-protein interactions promoting tumorigenesis. FAK seems also to have a role in the proliferation of tumor cells by its biochemical and biological association to cytoplasmic kinases such as src and ERK. However, the mechanisms that involve FAK in those processes are not yet clarify. The best characterized FAK phosporylation event is the auto-phosphorylation at the tyrosine397 (Tyr397) which creates a motif that is recognized by various SH2 domain-containing proteins, such as src which phosphorylates FAK on Tyr576. The c-terminal domain of FAK undergoes to several serine-phosphorylation events whose role is not well-understood. Previous studies in neural and endothelial cells (EC) have shown a new role for FAK in the regulation of centrosome functions during mitosis dependent on Ser732 phosphorylation. We have found that EGF stimulation of selected starved melanoma, thyroid and high stage ovarian tumor cells induced, in dividing cells, accumulation of phosphorylated (P-)FAK on Ser732. Inhibition of cell/substrate adhesion with specific anti-integrin antibodies demonstrated that P-FAKSer732 activation is not induced by integrin clustering. Confocal immunofluorescence and immunoprecipitation showed that, in these cells, P-FAKSer732 is localized in a perinuclear site independently from FA and P-FAKTyr397. Treatment of an high proliferative melanoma cell line with the Rock inhibitor Y27632 partially decreased the levels of P-FAKSer732, the cellular proliferation and impaired the mitotic spindle formation. FACS analysis showed that the P-FAKSer732 decreased upon treatment with the MEK inhibitor UO126 in a dose-dependent manner together with the levels of P(Ser 10) -Histone H3 thus demonstrating that FAK is involved in the mitotic process regulated by MEK/ERK activation. Indeed, P-FAKSer732 co-localized with microtubules of the mitotic spindle in proliferating cells and immunoprecipitated with the motor protein dynein and with the acetylated tubulin of polymerized microtubules. The finding of a novel role for FAK in tumor cells might provide the underpinning for therapeutic strategies in selected solid tumors. Partially supported by Italian Ministery of Health and Associazione Italiana per la Ricerca sul Cancro (AIRC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 226. doi:1538-7445.AM2012-226

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln--Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.