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The safety and activity of the multikinase inhibitor sorafenib were investigated in patients with relapsed or refractory lymphoproliferative disorders who received sorafenib (400 mg) twice daily until disease progression or appearance of significant clinical toxicity. The primary endpoint was overall response rate ( ORR). Biomarkers of sorafenib activity were analysed at baseline and during treatment. Thirty patients (median age, 61 years; range, 18-74) received a median of 4 months of therapy. Grade 3-4 toxicities included hand/foot skin reactions (20%), infections (12%), neutropenia (20%) and thrombocytopenia (14%). Two patients achieved complete remission ( CR), and two achieved partial remission ( PR) for an ORR of 13%. Stable disease ( SD) and progressive disease ( PD) was observed in 15 (50%) and 11 patients (37%), respectively. The median overall survival ( OS) for all patients was 16 months. For patients who achieved CR, PR and SD, the median time to progression and OS was 5 and 24 months, respectively. Compared with patients with PD, responsive patients had significantly higher baseline levels of extracellular signal-regulated kinase phosphorylation and autophagy and presented a significant reduction of these parameters after 1 month of therapy. Sorafenib was well tolerated and had a clinical activity that warrants development of combination regimens. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) and PI3K pathways are regulated by extensive crosstalk, occurring at different levels. In tumors, transactivation of the alternate pathway is a frequent 'escape' mechanism, suggesting that combined inhibition of both pathways may achieve synergistic antitumor activity. Here we show that, in the M14 melanoma model, simultaneous inhibition of both MEK and mammalian target of rapamycin (mTOR) achieves synergistic effects at suboptimal concentrations, but becomes frankly antagonistic in the presence of relatively high concentrations of MEK inhibitors. This observation led to the identification of a novel crosstalk mechanism, by which either pharmacologic or genetic inhibition of constitutive MEK signaling restores phosphatase and tensin homolog (PTEN) expression, both in vitro and in vivo, and inhibits downstream signaling through AKT and mTOR, thus bypassing the need for double pathway blockade. This appears to be a general regulatory mechanism and is mediated by multiple mechanisms, such as MAPK-dependent c-Jun and miR-25 regulation. Finally, PTEN upregulation appears to be a major effector of MEK inhibitors' antitumor activity, as cancer cells in which PTEN is inactivated are consistently more resistant to the growth inhibitory and anti-angiogenic effects of MEK blockade. [ABSTRACT FROM AUTHOR]

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors. [ABSTRACT FROM AUTHOR]

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42–Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42–Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells. [ABSTRACT FROM AUTHOR]

To evaluate the mutational profiles associated with BRAF mutations in human melanoma, we have studied BRAF, RAS, PTEN, TP53, CDKN2A and CDK4 genes and their expression in melanoma lesions. Owing to the lack of sufficient material from fresh specimens, we employed short-term cell lines obtained from melanoma biopsies. In all, 41 melanoma obtained from eight primary lesions, 20 nodal, 11 cutaneous and two visceral metastases from patients with sporadic (n=31), familial (n=4) and multiple melanoma (n=2) were analysed. The results revealed novel missense mutations in the BRAF, PTEN, CDKN2A and CDK4 genes. Overall, activating mutations of BRAF and loss of functional p16 and ARF were detected in the majority of melanomas (29/41, 36/41 and 29/41, respectively), while PTEN alterations/loss, NRAS and TP53 mutations occurred less frequently (6/41, 6/41 and 10/41, respectively). In the resulting 12 mutational profiles, p16/ARF loss associated with mutated BRAFV599E was the most represented (n=15). In addition, TP53 and PTEN mutations were always accompanied with BRAF alterations, while PTEN loss was found in association with CDKN2A or TP53 mutations in the absence of BRAF activation. The p16/ARF?+BRAF/RAS profile was significantly associated with a longer survival, while complex mutational profiles were detected in highly aggressive disease and poor survival. These data support the existence of several molecularly defined melanoma groups which likely reflect different clinical/biological behaviour, thus suggesting that a more extensive molecular classification of melanoma would significantly impact its clinical management.Oncogene (2004) 23, 5968-5977. doi:10.1038/sj.onc.1207780 Published online 14 June 2004 [ABSTRACT FROM AUTHOR]

Evaluates the frequency at which different antigens are seen by T cells of melanoma patients and healthy donors by human leukocyte antigen and peptide tetramer technology which stains T cells bearing the specific receptor for a given epitope. Characteristics of melanoma antigen proteins; Description of the microenvironment of melanoma lesions; Mechanisms of low T-cell responsiveness against tumor cells; Mechanism for tumor cells to shut down antitumor T-cell reactivity.

Many human tumor-associated antigens (TAAs) have recently been identified and molecularly characterized. When bound to major histocompatibility complex molecules, TAA peptides are recognized by T cells. Clinical studies have therefore been initiated to assess the therapeutic potential of active immunization or vaccination with TAA peptides in patients with metastatic cancer. So far, only a limited number of TAA peptides, mostly those recognized by CD8(+) T cells in melanoma patients, have been clinically tested. In some clinical trials, partial or complete tumor regression was observed in approximately 10%-30% of patients. No serious side effects have been reported. The clinical responses, however, were often not associated with a detectable T-cell-specific antitumor immune response when patients' T cells were evaluated in ex vivo assays. In this review, we analyze the available human TAA peptides, the potential immunogenicity (i.e., the ability to trigger a tumor-specific T-cell response) of TAA peptides in vitro and ex vivo, and the potential to construct slightly modified forms of TAA peptides that have increased T-cell stimulatory activity. We discuss the available data from clinical trials of TAA peptide-based vaccination (including those that used dendritic cells to present TAA peptides), identify possible reasons for the limited clinical efficacy of these vaccines, and suggest ways to improve the clinical outcome of TAA peptide-based vaccination for cancer patients. [ABSTRACT FROM AUTHOR]

A phase I clinical trial using autologous, IL-7 gene-modified tumor cells in patients with disseminated melanoma has been recently completed. Although no major clinical responses were observed, increased antitumor cytotoxicity was measured in postvaccine peripheral blood lymphocytes in a subset of treated patients. To analyze the in situ immune response, the T cell receptor β-chain variable region (BV) repertoire of T cells infiltrating postvaccine lesions was studied in two patients, and compared with that of T cells present in prevaccine ones, in peripheral blood lymphocytes, and, in one patient, in delayed type hypersensitivity (DTH) sites of autologous melanoma inoculum. A relative expansion of T cells expressing few BVs was observed in all postvaccine metastases, and their intratumoral presence was confirmed by immunohistochemistry. Length pattern analysis of the complementarity determining region 3 (CDR3) indicated that the repertoire of T cells expressing some of these BVs was heterogeneous. At difference, CDR3, β-chain joining region usage, and sequence analysis enabled us to demonstrate, within a T-cell subpopulation commonly expanded at DTH sites and at the postvaccine lesion of patient 1, that both DTH sites contained identical dominant T-cell clonotypes. One of them was also expressed at increased relative frequency in the postvaccine lesion compared to prevaccine specimens. These results provide evidence for immunological changes, including in situ clonally expanded T cells, in metastases of patients vaccinated with IL-7 gene-transduced cells. [ABSTRACT FROM AUTHOR]

Summary We previously reported that a melanoma antigen, recognized by tumor-specific cytotoxic T lymphocytes, was encoded by intron sequences retained in a partially spliced transcript of the tyrosinase-related protein-2/DOPAchrome tautomerase gene. At difference with the mRNA encoding tyrosinase-related protein-2, this anomalous transcript was not expressed in melanocytes. This study examined whether neoplastic and/or normal cells of the melanocytic lineage could express additional forms of tyrosinase-related protein-2 mRNA. Screening of a melanoma-derived cDNA library with a tyrosinase-related protein-2 probe allowed identification of two novel isoforms. The first, tyrosinase-related protein-2-long tail, corresponds to the dominant transcript detected on melanomas and melanocytes by northern blot analysis. Tyrosinase-related protein-2-long tail is identical to the tyrosinase-related protein-2-encoding published cDNA sequence except for an extended 3′-untranslated region and is originated by alternative polyadenylation. This novel 3′-untranslated region contains an alternatively spliced, tyrosinase-related protein-2 last exon in the second isoform (tyrosinase-related protein-2–8b). The protein encoded by tyrosinase-related protein-2–8b is identical to tyrosinase-related protein-2 in its first 460 amino acids but possesses a different carboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein-2-long tail exhibited DOPA-chrome tautomerase activity, when transiently transfected into COS-7 cells. On the contrary, no detectable activity was exhibited by tyrosinase-related protein-2–8b. Reverse transcription–polymerase chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2–8b are expressed by tyrosinase-related protein-2-positive melanomas and normal melanocytes. Moreover all cell lines positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the human tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield different isoforms by alternative poly(A) site usage or by alternative splicing. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis. [ABSTRACT FROM AUTHOR]

High-grade Gastroenteropancreatic Neuroendocrine neoplasms (H-NENs) comprehend well-differentiated tumors (NET G3) and poorly differentiated carcinomas (NEC) with proliferative activity indexes as mitotic count (MC) >20 mitoses/10 HPF and Ki-67 >20%. At present, no specific therapy for H-NENs exists and the several evidences of microenvironment involvement in their pathogenesis pave the way for tailored therapies. Forty-five consecutive cases, with available information about T-cell, immune, and non-immune markers, from surgical pathology and clinical databases of 2 Italian institutions were immunostained for Arginase, CD33, CD163 and CD66 myeloid markers. The association between features was assessed by Spearman's correlation coefficient. A unsupervised K-means algorithm was used to identify clusters of patients according to inputs of microenvironment features and the relationship between clusters and clinicopathological features, including cancer-specific survival (CSS), was analyzed. The H-NEN population was composed of 6 (13.3%) NET G3 and 39 (86.7%) NEC. Overall, significant positive associations were found between myeloid (CD33, CD163 and Arginase) and T/immune markers (CD3, CD4, CD8, PD-1 and HLA-I). Myeloid and T-cell markers CD3 and CD8 identified two clusters of patients from unsupervised K-means analysis. Cases grouped in cluster 1 with more myeloid infiltrates, T cell, HLA and expression of inhibitory receptors and ligands in the stroma (PD-1, PD-L1) had significantly better CSS than patients in cluster 2. Multivariable analysis showed that Ki-67 (>55 vs. <55, HR 8.60, CI 95% 2.61–28.33, p < 0.0001) and cluster (1 vs. 2, HR 0.43, CI 95% 0.20–0.93, p = 0.03) were significantly associated with survival. High grade gastroenteropancreatic neuroendocrine neoplasms can be further classified into two prognostic sub-populations of tumors driven by different tumor microenvironments and immune features able to generate the framework for evaluating new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Almost 25% of lung cancers (LCs) occur in never-smokers. LC inflammatory profile, based on plasma C-reactive protein levels (CRP), predicts mortality, independently by smoking-status. We hypothesized that: CRP could be associated with tumor immune contexture (TIC) in never-smokers and both these two parameters may improve their prognosis. Sixty-eight never-smokers LC patients with high or low CRP were selected. The programmed cell death protein 1 (PD-1) and its ligand (PD-L1), the human leukocyte antigens (HLA-DR and HLA-I), CD8, CD4, CD3, CD33, CD163, and CD68 were evaluated by immunohistochemistry on surgical samples given TIC evaluation. The classification model based on TIC scores was generated by Classification and Regression Tree analysis. Tumor mutational burden was evaluated by targeted next-generation sequencing. Exclusively high CRP (H-CRP) subset showed PD-L1 expression in 35% of LC as well as lower HLA-I and HLA-DR in their stromal cells. CD3, CD4, CD8, HLA-I, HLA-DR tumor cells staining were associated with a "low inflammatory profile" subset. CRP and LC immune profiles drive clinical outcome: 5-year survival 88% against 8% was associated with low and high-risk profiles (p < 0.0001). Clinical outcome prediction in never-smoker LC patients may be improved by both CRP and tumor immune contexture evaluation. [ABSTRACT FROM AUTHOR]

Thyroid carcinoma (TC) comprises several histotypes with different aggressiveness, from well (papillary carcinoma, PTC) to less differentiated forms (poorly differentiated and anaplastic thyroid carcinoma, PDTC and ATC, respectively). Previous reports have suggested a functional role for cancer-associated fibroblasts (CAFs) or senescent TC cells in the progression of PTC. In this study, we investigated the presence of CAFs and senescent cells in proprietary human TCs including PTC, PDTC, and ATC. Screening for the driving lesions BRAFV600E and N/H/KRAS mutations, and gene fusions was also performed to correlate results with tumor genotype. In samples with unidentified drivers, transcriptomic profiles were used to establish a BRAF- or RAS-like molecular subtype based on a gene signature derived from The Cancer Genome Atlas. By using immunohistochemistry, we found co-occurrence of stromal CAFs and senescent TC cells at the tumor invasive front, where deposition of collagen (COL1A1) and expression of lysyl oxidase (LOX) enzyme were also detected, in association with features of local invasion. Concurrent high expression of CAFs and of the senescent TC cells markers, COL1A1 and LOX was confirmed in different TC histotypes in proprietary and public gene sets derived from Gene Expression Omnibus (GEO) repository, and especially in BRAF mutated or BRAF-like tumors. In this study, we show that CAFs and senescent TC cells co-occur in various histotypes of BRAF-driven thyroid tumors and localize at the tumor invasive front. [ABSTRACT FROM AUTHOR]

Background: Melanoma, the most aggressive form of skin cancer, is characterized by high rates of metastasis, drug resistance and mortality. Here we investigated the role of Semaphorin 5A (Sema5A) on the properties associated with melanoma progression and the factors involved in Sema5A regulation. Methods: Western blotting, qRT-PCR, Chromatin immunoprecipitation (ChIP) assay, immunohistochemistry of melanoma patient specimens and xenograft tissues, in vitro Transwell assay for cell migration and invasion evaluation, in vitro capillary-like structure formation analysis. Results: A significant correlation of Sema5A mRNA expression and melanoma progression was observed by analyzing GEO profile dataset. Endogenous Sema5A protein was detected in 95% of human melanoma cell lines tested, in 70% of metastatic specimens from patients affected by melanoma, and 16% of in situ melanoma specimens showed a focal positivity. We demonstrated that Sema5A regulates in vitro cell migration and invasion and the formation of vasculogenic structures. We also found an increase of Sema5A at both mRNA and protein level after forced expression of Bcl-2. By use of transcriptional and proteasome inhibitors, we showed that Bcl-2 increases the stability of Sema5A mRNA and protein. Moreover, by ChIP we demonstrated that Sema5A expression is under the control of the transcription factor c-Myb and that c-Myb recruitment on Sema5A promoter is increased after Bcl-2 overexpression. Finally, a concomitant decrease in the expression of Sema5A, Bcl-2 and c-Myb proteins was observed in melanoma cells after miR-204 overexpression. Conclusion: Overall our data provide evidences supporting the role of Sema5A in melanoma progression and the involvement of Bcl-2, miR-204 and c-Myb in regulating its expression. [ABSTRACT FROM AUTHOR]

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots., Methods and Results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo , triggering ADCC by healthy donors' and patients' peripheral blood cells., Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Muraro, Montico, Lum, Colizzi, Giurato, Salvati, Guerrieri, Rizzo, Comaro, Canzonieri, Anichini, Del Vecchio, Mortarini, Milione, Weisz, Pizzichetta, Simpson, Dolcetti, Fratta and Sigalotti.)

Background: Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor, that is refractory to standard treatment and to immunotherapy with immune-checkpoint inhibitors (ICI). Noteworthy, melanoma brain metastases (MM-BM), that share the same niche as GBM, frequently respond to current ICI therapies. Epigenetic modifications regulate GBM cellular proliferation, invasion, and prognosis and may negatively regulate the cross-talk between malignant cells and immune cells in the tumor milieu, likely contributing to limit the efficacy of ICI therapy of GBM. Thus, manipulating the tumor epigenome can be considered a therapeutic opportunity in GBM., Methods: Microarray transcriptional and methylation profiles, followed by gene set enrichment and IPA analyses, were performed to study the differences in the constitutive expression profiles of GBM vs MM-BM cells, compared to the extracranial MM cells and to investigate the modulatory effects of the DNA hypomethylating agent (DHA) guadecitabine among the different tumor cells. The prognostic relevance of DHA-modulated genes was tested by Cox analysis in a TCGA GBM patients' cohort., Results: The most striking differences between GBM and MM-BM cells were found to be the enrichment of biological processes associated with tumor growth, invasion, and extravasation with the inhibition of MHC class II antigen processing/presentation in GBM cells. Treatment with guadecitabine reduced these biological differences, shaping GBM cells towards a more immunogenic phenotype. Indeed, in GBM cells, promoter hypomethylation by guadecitabine led to the up-regulation of genes mainly associated with activation, proliferation, and migration of T and B cells and with MHC class II antigen processing/presentation. Among DHA-modulated genes in GBM, 7.6% showed a significant prognostic relevance. Moreover, a large set of immune-related upstream-regulators (URs) were commonly modulated by DHA in GBM, MM-BM, and MM cells: DHA-activated URs enriched for biological processes mainly involved in the regulation of cytokines and chemokines production, inflammatory response, and in Type I/II/III IFN-mediated signaling; conversely, DHA-inhibited URs were involved in metabolic and proliferative pathways., Conclusions: Epigenetic remodeling by guadecitabine represents a promising strategy to increase the efficacy of cancer immunotherapy of GBM, supporting the rationale to develop new epigenetic-based immunotherapeutic approaches for the treatment of this still highly deadly disease., (© 2024. The Author(s).)

Association with hypomethylating agents is a promising strategy to improve the efficacy of immune checkpoint inhibitors-based therapy. The NIBIT-M4 was a phase Ib, dose-escalation trial in patients with advanced melanoma of the hypomethylating agent guadecitabine combined with the anti-CTLA-4 antibody ipilimumab that followed a traditional 3 + 3 design (NCT02608437). Patients received guadecitabine 30, 45 or 60 mg/m 2 /day subcutaneously on days 1 to 5 every 3 weeks starting on week 0 for a total of four cycles, and ipilimumab 3 mg/kg intravenously starting on day 1 of week 1 every 3 weeks for a total of four cycles. Primary outcomes of safety, tolerability, and maximum tolerated dose of treatment were previously reported. Here we report the 5-year clinical outcome for the secondary endpoints of overall survival, progression free survival, and duration of response, and an exploratory integrated multi-omics analysis on pre- and on-treatment tumor biopsies. With a minimum follow-up of 45 months, the 5-year overall survival rate was 28.9% and the median duration of response was 20.6 months. Re-expression of immuno-modulatory endogenous retroviruses and of other repetitive elements, and a mechanistic signature of guadecitabine are associated with response. Integration of a genetic immunoediting index with an adaptive immunity signature stratifies patients/lesions into four distinct subsets and discriminates 5-year overall survival and progression free survival. These results suggest that coupling genetic immunoediting with activation of adaptive immunity is a relevant requisite for achieving long term clinical benefit by epigenetic immunomodulation in advanced melanoma patients., (© 2023. Springer Nature Limited.)

Background: Chemoimmunotherapy represents the standard of care for patients with advanced non-small cell lung cancer (NSCLC) and programmed death-ligand 1 (PD-L1) <50%. Although single-agent pembrolizumab has also demonstrated some activity in this setting, no reliable biomarkers yet exist for selecting patients likely to respond to single-agent immunotherapy. The main purpose of the study was to identify potential new biomarkers associated with progression-free-survival (PFS) within a multiomics analysis., Methods: PEOPLE (NTC03447678) was a prospective phase II trial evaluating first-line pembrolizumab in patients with advanced EGFR and ALK wild type treatment-naïve NSCLC with PD-L1 <50%. Circulating immune profiling was performed by determination of absolute cell counts with multiparametric flow cytometry on freshly isolated whole blood samples at baseline and at first radiological evaluation. Gene expression profiling was performed using nCounter PanCancer IO 360 Panel (NanoString) on baseline tissue. Gut bacterial taxonomic abundance was obtained by shotgun metagenomic sequencing of stool samples at baseline. Omics data were analyzed with sequential univariate Cox proportional hazards regression predicting PFS, with Benjamini-Hochberg multiple comparisons correction. Biological features significant with univariate analysis were analyzed with multivariate least absolute shrinkage and selection operator (LASSO)., Results: From May 2018 to October 2020, 65 patients were enrolled. Median follow-up and PFS were 26.4 and 2.9 months, respectively. LASSO integration analysis, with an optimal lambda of 0.28, showed that peripheral blood natural killer cells/CD56dimCD16+ (HR 0.56, 0.41-0.76, p=0.006) abundance at baseline and non-classical CD14dimCD16+monocytes (HR 0.52, 0.36-0.75, p=0.004), eosinophils (CD15+CD16-) (HR 0.62, 0.44-0.89, p=0.03) and lymphocytes (HR 0.32, 0.19-0.56, p=0.001) after first radiologic evaluation correlated with favorable PFS as well as high baseline expression levels of CD244 (HR 0.74, 0.62-0.87, p=0.05) protein tyrosine phosphatase receptor type C (HR 0.55, 0.38-0.81, p=0.098) and killer cell lectin like receptor B1 (HR 0.76, 0.66-0.89, p=0.05). Interferon-responsive factor 9 and cartilage oligomeric matrix protein genes correlated with unfavorable PFS (HR 3.03, 1.52-6.02, p 0.08 and HR 1.22, 1.08-1.37, p=0.06, corrected). No microbiome features were selected., Conclusions: This multiomics approach was able to identify immune cell subsets and expression levels of genes associated to PFS in patients with PD-L1 <50% NSCLC treated with first-line pembrolizumab. These preliminary data will be confirmed in the larger multicentric international I3LUNG trial (NCT05537922)., Trial Registration Number: 2017-002841-31., Competing Interests: Competing interests: GLR provided consultation, attended advisory boards and/or provided lectures for the following organizations, from whom received honoraria or education grants: Merck Sharp and Dohme, Takeda, Amgen, Eli Lilly, BMS, Roche, Italfarmaco, Novartis, Sanofi, Pfizer and AstraZeneca. AP declares personal fees from AstraZeneca, Italfarmaco, Roche, BMS. RF declares advisory role from Merck Sharp and Dohme. FDB provided consultation, attended advisory boards and/or provided lectures for the following organizations, from whom received honoraria or education grants: Amgen, AstraZeneca, Boehringer-Ingelheim, BMS, Eli Lilly, F. Hoffmann-La Roche, Ignyta, Merck Sharp and Dohme, Merck Serono, Novartis, Pfizer.CP declares personal fees from Italfarmaco, AstraZeneca, BMS and Merck Sharp and Dohme.MCG declares personal financial interests with the following organizations: AstraZeneca, MSD International GmbH, BMS, Boehringer Ingelheim Italia S.p.A, Celgene, Eli Lilly, Ignyta, Incyte, Inivata, MedImmune, Novartis, Pfizer, Roche, Takeda, Seattle Genetics, Mirati, Daiichi Sankyo, Regeneron, Merck, Ose Immuno Therapeutics, Blueprint, Jansenn, Sanofi; she also declares Institutional financial interests with the following organizations: Eli Lilly, MSD, Pfizer (MISP); AstraZeneca, MSD International GmbH, BMS, Boehringer Ingelheim Italia S.p.A, Celgene, Eli Lilly, Ignyta, Incyte, MedImmune, Novartis, Pfizer, Roche, Takeda, Tiziana, Foundation Medicine, Glaxo Smith Kline GSK, Spectrum pharmaceuticals., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents., Methods: Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets., Results: Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset., Conclusions: The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches., (© 2022. The Author(s).)

We previously published the results of a pilot study showing that vaccination with tumor-loaded dendritic cells (DCs) induced both T and B cell response and produced clinical benefit in the absence of toxicity in patients with relapsed, indolent non-Hodgkin lymphoma (iNHL). The purpose of the present short report is to provide a 15-year follow-up of our study and to expand the biomarker analysis previously performed. The long-term follow-up highlighted the absence of particular or delayed toxicity and the benefit of active immunization with DCs loaded with autologous, heat-shocked and UV-C treated tumor cells in relapsed iNHL (5-year and 10-year progression-free survival (PFS) rates: 55.6% and 33.3%, respectively; 10-year overall survival (OS) rate: 83.3%). Female patients experienced a better PFS (p=0.016) and a trend towards a better OS (p=0.185) compared with male patients. Of note, we observed a non-negligible fraction of patients (22%) who experienced a long-lasting complete response. In a targeted gene expression profiling of pre-treatment tumor biopsies in 11 patients with available formalin-fixed, paraffin-embedded tissue, we observed that KIT , ATG12 , TNFRSF10C , PBK , ITGA2 , GATA3 , CLU , NCAM1 , SYT17 and LTK were differentially expressed in patients with responder versus non-responder tumors. The characterization of peripheral monocytic cells in a subgroup of 14 patients with available baseline blood samples showed a higher frequency of the subset of CD14 ++ CD16 + cells (intermediate monocytes) in patients with responding tumors. Since in patients with relapsed iNHL the available therapeutic options are often incapable of inducing a long-lasting complete remission and can be sometimes characterized by intolerable toxicity, we think that the encouraging results of our long-term follow-up analysis represent a stimulus to further investigate the role of active vaccination in this specific setting and in earlier lines of therapy and to explore novel combinatorial strategies encompassing other innovative immunotherapy agents, such as immune-checkpoint inhibitors., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80 hi CD115 hi C3aR hi CD88 hi ), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80 + HO-1 + bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80 + HO-1 + TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1 + myeloid subgroup as a new antimetastatic target and prognostic blood marker.

Development of strong immune evasion has been traditionally associated with the late stages of solid tumor progression, since advanced cancers are more likely to have reached the third phase of the immunoediting process. However, by integrating a variety of approaches, evidence for active immune escape mechanisms has been found even in the pre-invasive lesions that later progress to the main NSCLC histotypes. Pre-invasive lesions of adenocarcinoma (LUAD) and of squamous cell carcinoma (LUSC) can show impaired antigen presentation, loss of heterozygosity at the Human Leukocyte Antigen (HLA) region, neoantigen silencing, activation of immune checkpoints, altered TH1/TH2 cytokine ratios, and immune contexture evolution. Analysis of large panels of LUAD vs. LUSC, of early stage NSCLC vs. normal lung tissue, of specific molecular subsets of NSCLC, and of distinct regions within the same tumor, indicates that all these processes of immune escape continue to evolve in the invasive stage of NSCLC, are associated with inter- and intra-tumor heterogeneity, and contribute to resistance to therapy by immune checkpoint blockade (ICB). In this review, we will discuss the most recent evidence on immune escape mechanisms developing from the precursor to invasive stage in NSCLC, and the contribution of immune evasion to resistance to ICB in lung cancer.

Background: Atypical melanocytic tumors (AMTs) include a wide spectrum of melanocytic neoplasms that represent a challenge for clinicians due to the lack of a definitive diagnosis and the related uncertainty about their management. This study analyzed clinicopathologic features and sentinel node status as potential prognostic factors in patients with AMTs., Patients and Methods: Clinicopathologic and follow-up data of 238 children, adolescents, and adults with histologically proved AMTs consecutively treated at 12 European centers from 2000 through 2010 were retrieved from prospectively maintained databases. The binary association between all investigated covariates was studied by evaluating the Spearman correlation coefficients, and the association between progression-free survival and all investigated covariates was evaluated using univariable Cox models. The overall survival and progression-free survival curves were established using the Kaplan-Meier method., Results: Median follow-up was 126 months (interquartile range, 104-157 months). All patients received an initial diagnostic biopsy followed by wide (1 cm) excision. Sentinel node biopsy was performed in 139 patients (58.4%), 37 (26.6%) of whom had sentinel node positivity. There were 4 local recurrences, 43 regional relapses, and 8 distant metastases as first events. Six patients (2.5%) died of disease progression. Five patients who were sentinel node-negative and 3 patients who were sentinel node-positive developed distant metastases. Ten-year overall and progression-free survival rates were 97% (95% CI, 94.9%-99.2%) and 82.2% (95% CI, 77.3%-87.3%), respectively. Age, mitotic rate/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss were factors affecting prognosis in the whole series and the sentinel node biopsy subgroup., Conclusions: Age >20 years, mitotic rate >4/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss proved to be worse prognostic factors in patients with ATMs. Sentinel node status was not a clear prognostic predictor.

The potential immune intersection between COVID-19 disease and cancer therapy raises important practical clinical questions and highlights multiple scientific gaps to be filled. Among available therapeutic approaches to be considered, immune checkpoint inhibitors (ICI) seem to require major attention as they may act at the crossroads between cancer treatment and COVID-19 disease, due to their profound immunomodulatory activity. On the basis of available literature evidence, we suggest guidance to consider for treating physicians, and propose areas of clinical and preclinical investigation. Comprehensively, although with the necessary caution, ICI therapy seems to remain a suitable therapeutic option for patients with cancer during the COVID-19 pandemic., (©2020 American Association for Cancer Research.)

Purpose: Thin melanomas (T1; ≤ 1 mm) constitute 70% of newly diagnosed cutaneous melanomas. Regional node metastasis determined by sentinel node biopsy (SNB) is an important prognostic factor for T1 melanoma. However, current melanoma guidelines do not provide clear indications on when to perform SNB in T1 disease and stress an individualized approach to SNB that considers all clinicopathologic risk factors. We aimed to identify determinants of sentinel node (SN) status for incorporation into an externally validated nomogram to better select patients with T1 disease for SNB., Patients and Methods: The development cohort comprised 3,666 patients with T1 disease consecutively treated at the Istituto Nazionale Tumori (Milan, Italy) between 2001 and 2018; 4,227 patients with T1 disease treated at 13 other European centers over the same period formed the validation cohort. A random forest procedure was applied to the development data set to select characteristics associated with SN status for inclusion in a multiple binary logistic model from which a nomogram was elaborated. Decision curve analyses assessed the clinical utility of the nomogram., Results: Of patients in the development cohort, 1,635 underwent SNB; 108 patients (6.6%) were SN positive. By univariable analysis, age, growth phase, Breslow thickness, ulceration, mitotic rate, regression, and lymphovascular invasion were significantly associated with SN status. The random forest procedure selected 6 variables (not growth phase) for inclusion in the logistic model and nomogram. The nomogram proved well calibrated and had good discriminative ability in both cohorts. Decision curve analyses revealed the superior net benefit of the nomogram compared with each individual variable included in it as well as with variables suggested by current guidelines., Conclusion: We propose the nomogram as a decision aid in all patients with T1 melanoma being considered for SNB.

Purpose: The immunomodulatory activity of DNA hypomethylating agents (DHAs) suggests they may improve the effectiveness of cancer immunotherapies. The phase Ib NIBIT-M4 trial tested this hypothesis using the next-generation DHA guadecitabine combined with ipilimumab., Patients and Methods: Patients with unresectable stage III/IV melanoma received escalating doses of guadecitabine 30, 45, or 60 mg/m 2 /day subcutaneously on days 1 to 5 every 3 weeks, and ipilimumab 3 mg/kg intravenously on day 1 every 3 weeks, starting 1 week after guadecitabine, for four cycles. Primary endpoints were safety, tolerability, and MTD of treatment; secondary were immune-related (ir) disease control rate (DCR) and objective response rate (ORR); and exploratory were changes in methylome, transcriptome, and immune contextures in sequential tumor biopsies, and pharmacokinetics., Results: Nineteen patients were treated; 84% had grade 3/4 adverse events, and neither dose-limiting toxicities per protocol nor overlapping toxicities were observed. Ir-DCR and ir-ORR were 42% and 26%, respectively. Median CpG site methylation of tumor samples ( n = 8) at week 4 (74.5%) and week 12 (75.5%) was significantly ( P < 0.05) lower than at baseline (80.3%), with a median of 2,454 (week 4) and 4,131 (week 12) differentially expressed genes. Among the 136 pathways significantly ( P < 0.05; Z score >2 or ←2) modulated by treatment, the most frequently activated were immune-related. Tumor immune contexture analysis ( n = 11) demonstrated upregulation of HLA class I on melanoma cells, an increase in CD8 + , PD-1 + T cells and in CD20 + B cells in posttreatment tumor cores., Conclusions: Treatment of guadecitabine combined with ipilimumab is safe and tolerable in advanced melanoma and has promising immunomodulatory and antitumor activity., (©2019 American Association for Cancer Research.)

T-cell-based immunotherapy strategies have profoundly improved the clinical management of several solid tumors and hematological malignancies. A recently developed and promising immunotherapy approach is to redirect polyclonal MHC-unrestricted T lymphocytes toward cancer cells by bispecific antibodies (bsAbs) that engage the CD3 complex and a tumor-associated antigen (TAA). The TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) is an attractive immunotherapy target, frequently expressed by neoplastic cells, that we decided to exploit as a TAA. We found that a TRAIL-R2xCD3 bsAb efficiently activates T cells and specifically redirect their cytotoxicity against cancer cells of different origins in vitro , thereby demonstrating its potential as a pan-carcinoma reagent. Moreover, to mimic in vivo conditions, we assessed its ability to retarget T-cell activity in an ex vivo model of ovarian cancer patients' ascitic fluids containing both effector and target cells-albeit with a suboptimal effector-to-target ratio-with remarkable results., (Copyright © 2019 Satta, Grazia, Caroli, Frigerio, Di Nicola, Raspagliesi, Mezzanzanica, Zaffaroni, Gianni, Anichini and Figini.)

Microenvironment-related immune and inflammatory markers, when combined with established Ki-67 and morphology parameters, can improve prognostic prediction in gastro-entero-pancreatic neuroendocrine neoplasms (GEP-NENs). Therefore, we evaluated the prognostic value of microenvironment and tumor inflammatory features (MoTIFs) in GEP-NENs. For this purpose, formalin-fixed paraffin-embedded tissue sections from 350 patients were profiled by immunohistochemistry for immune, inflammatory, angiogenesis, proliferation, NEN-, and fibroblast-related markers. A total of 314 patients were used to generate overall survival (OS) and disease-free survival (DFS) MoTIFs prognostic indices (PIs). PIs and additional variables were assessed using Cox models to generate nomograms for predicting 5-year OS and DFS. A total of 36 patients were used for external validation of PIs and nomograms' prognostic segregations. From our analysis, G1/G2 versus G3 GEP-NENs showed phenotypic divergence with immune-inflammatory markers. HLA, CD3, CD8, and PD-1/PD-L1 IHC expression separated G3 into two sub-categories with high versus low adaptive immunity-related features. MoTIFs PI for OS based on COX-2 Tumor(T) > 4, PD-1 Stromal(S) > 0, CD8 S < 1, and HLA-I S < 1 was associated with worst survival (hazard ratio [HR] 2.50; 95% confidence interval [CI], 2.12-2.96; p < 0.0001). MoTIFs PI for DFS was based on COX-2 T > 4, PD-1 S > 4, HLA-I S < 1, HLA-I T < 2, HLA-DR S < 6 (HR 1.77; 95% CI, 1.58-1.99; p < 0.0001). Two nomograms were developed including morphology (HR 4.83; 95% CI, 2.30-10.15; p < 0.001) and Ki-67 (HR 11.32; 95% CI, 5.28-24.24; p < 0.001) for OS, and morphology (PI = 0: HR 10.23; 95% CI, 5.67-18.47; PI = 5: HR 2.87; 95% CI, 1.21-6.81; p < 0.001) and MoTIFs PI for DFS in well-differentiated GEP-NENs (HR 6.21; 95% CI, 2.52-13.31; p < 0.001). We conclude that G1/G2 to G3 transition is associated with immune-inflammatory profile changes; in fact, MoTIFs combined with morphology and Ki-67 improve 5-year DFS prediction in GEP-NENs. The immune context of a subset of G3 poorly differentiated tumors is consistent with activation of adaptive immunity, suggesting a potential for responsiveness to immunotherapy targeting immune checkpoints., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Gene signatures are increasingly being used to infer the immune composition of the tumor microenvironment. This strategy holds the promise for earlier detection, identification of patients at higher risk of progression, and for understanding therapeutic response and resistance to immunotherapy. This gene signature approach is now being integrated with information from genomic changes, gene networks, and master immunoregulatory genes, and this development can lead to identify the main determinants shaping the tumor immune landscape. A study in this issue of Cancer Research indicates a way forward to discover prognostic immune-related subsets in primary melanoma and to understand major determinants impairing protective immunity in early stage of disease. See related article by Poźniak et al., p. 2684 ., (©2019 American Association for Cancer Research.)

Discovery of new actionable targets and functional networks in melanoma is an urgent need as only a fraction of metastatic patients achieves durable clinical benefit by targeted therapy or immunotherapy approaches. Here we show that NFATc2 expression is associated with an EMT-like transcriptional program and with an invasive melanoma phenotype, as shown by analysis of melanoma cell lines at the mRNA and protein levels, interrogation of the TCGA melanoma dataset and characterization of melanoma lesions by immunohistochemistry. Gene silencing or pharmacological inhibition of NFATc2 downregulated EMT-related genes and AXL, and suppressed c-Myc, FOXM1, and EZH2. Targeting of c-Myc suppressed FOXM1 and EZH2, while targeting of FOXM1 suppressed EZH2. Inhibition of c-Myc, or FOXM1, or EZH2 downregulated EMT-related gene expression, upregulated MITF and suppressed migratory and invasive activity of neoplastic cells. Stable silencing of NFATc2 impaired melanoma cell proliferation in vitro and tumor growth in vivo in SCID mice. In NFATc2 + EZH2 + melanoma cell lines pharmacological co-targeting of NFATc2 and EZH2 exerted strong anti-proliferative and pro-apoptotic activity, irrespective of BRAF or NRAS mutations and of BRAF inhibitor resistance. These results provide preclinical evidence for a role of NFATc2 in shaping the EMT-like melanoma phenotype and reveal a targetable vulnerability associated with NFATc2 and EZH2 expression in melanoma cells belonging to different mutational subsets.

Purpose: Hyperprogression (HP), a paradoxical boost in tumor growth, was described in a subset of patients treated with immune checkpoint inhibitors (ICI). Neither clinicopathologic features nor biological mechanisms associated with HP have been identified., Experimental Design: Among 187 patients with non-small cell lung cancer (NSCLC) treated with ICI at our institute, cases with HP were identified according to clinical and radiologic criteria. Baseline histologic samples from patients treated with ICI were evaluated by IHC for myeloid and lymphoid markers. T-cell-deficient mice, injected with human lung cancer cells and patient-derived xenografts (PDX) belonging to specific mutational subsets, were assessed for tumor growth after treatment with antibodies against mouse and human programmed death receptor-1 (PD-1). The immune microenvironment was evaluated by flow cytometry and IHC., Results: Among 187 patients, 152 were evaluable for clinical response. We identified four categories: 32 cases were defined as responders (21%), 42 patients with stable disease (27.7%), 39 cases were defined as progressors (25.7%), and 39 patients with HP (25.7%). Pretreatment tissue samples from all patients with HP showed tumor infiltration by M2-like CD163 + CD33 + PD-L1 + clustered epithelioid macrophages. Enrichment by tumor-associated macrophages (TAM) was observed, even in tumor nodules from immunodeficient mice injected with human lung cancer cells and with PDXs. In these models, tumor growth was enhanced by treatment with anti-PD-1 but not anti-PD-1 F(ab) 2 fragments., Conclusions: These results suggest a crucial role of TAM reprogramming, upon Fc receptor engagement by ICI, eventually inducing HP and provide clues on a distinctive immunophenotype potentially able to predict HP. See related commentary by Knorr and Ravetch, p. 904 ., (©2018 American Association for Cancer Research.)

Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or TRAIL-receptor agonistic monoclonal antibodies promote apoptosis in most cancer cells, and the differential expression of TRAIL-R2 between tumor and normal tissues allows its exploitation as a tumor-associated antigen. The use of these antibodies as anticancer agents has been extensively studied, but the results of clinical trials were disappointing. The observed lack of anticancer activity could be attributed to intrinsic or acquired resistance of tumor cells to this type of treatment. A possible strategy to circumvent drug resistance would be to strike tumor cells with a second modality based on a different mechanism of action. We therefore set out to generate and optimize a bispecific antibody targeting TRAIL-R2 and CD3. After the construction of different bispecific antibodies in tandem-scFv or single-chain diabody formats to reduce possible immunogenicity, we selected a humanized bispecific antibody with very low aggregates and long-term high stability and functionality. This antibody triggered TRAIL-R2 in an agonistic manner and its anticancer activity proved dramatically potentiated by the redirection of cytotoxic T cells against both sensitive and resistant melanoma cells. The results of our study show that combining the TRAIL-based antitumor strategy with an immunotherapeutic approach in a single molecule could be an effective addition to the anticancer armamentarium.

Advanced Merkel cell carcinoma (MCC) is a very aggressive, rare neuroendocrine tumor of the skin with a high frequency of locoregional recurrence and metastasis, and a high mortality rate. Surgical resection, sentinel lymph node biopsy, and radiotherapy represent the gold standard of treatment in patients with localized disease, while chemotherapy has a significant role in the treatment of advanced disease. However, no definitive evidence on the survival impact of radiotherapy in the advanced stages has been provided to date, and response to chemotherapy remains brief in the majority of cases, indicating an urgent need for alternative approaches. Biological and genome sequencing studies have implicated multiple molecular pathways in MCC, thus leading to the development of new agents that target angiogenic factors, anti-apoptosis molecules, poly-ADP ribose polymerase, intracellular signal proteins such as the PI3K/AKT/mTOR pathway, and peptide receptors such as somatostatin receptors. More recently, immunotherapy agents such as avelumab, pembrolizumab, and nivolumab, which act by blocking the programmed cell-death (PD)-1/PD-L1 immune checkpoint, have shown promising results, especially in the advanced setting, and should now be considered standard of care for metastatic MCC. Current research is focusing on developing new immunotherapeutic strategies, identifying predictive biomarker to aid in the selection of patients responsive to immunotherapy, and defining combination approaches to increase efficacy in refractory patients.

Immunotherapy of non-small cell lung cancer (NSCLC), by immune checkpoint inhibitors, has profoundly improved the clinical management of advanced disease. However, only a fraction of patients respond and no effective predictive factors have been defined. Here, we discuss the prospects for identification of such predictors of response to immunotherapy, by fostering an in-depth analysis of the immune landscape of NSCLC. The emerging picture, from several recent studies, is that the immune contexture of NSCLC lesions is a complex and heterogeneous feature, as documented by analysis for frequency, phenotype and spatial distribution of innate and adaptive immune cells, and by characterization of functional status of inhibitory receptor + T cells. The complexity of the immune landscape of NSCLC stems from the interaction of several factors, including tumor histology, molecular subtype, main oncogenic drivers, nonsynonymous mutational load, tumor aneuploidy, clonal heterogeneity and tumor evolution, as well as the process of epithelial-mesenchymal transition. All these factors contribute to shape NSCLC immune profiles that have clear prognostic significance. An integrated analysis of the immune and molecular profile of the neoplastic lesions may allow to define the potential predictive role of the immune landscape for response to immunotherapy.

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 (T EM ) 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 ., (©2016 American Association for Cancer Research.)

Despite the success of immune checkpoint blockade in melanoma, the majority of patients do not respond. We hypothesized that the T and NK cell subset frequencies and expression levels of their receptors may predict responses and clinical outcome of anti-CTLA-4 treatment. We thus characterized the NK and T cell phenotype, as well as serum levels of several cytokines in 67 melanoma patients recruited in Italy and Sweden, using samples drawn prior to and during treatment. Survival correlated with low expression of the inhibitory receptor TIM-3 on circulating T and NK cells prior to and during treatment and with the increased frequency of mature circulating NK cells (defined as CD3 - CD56 dim CD16 + ) during treatment. Survival also correlated with low levels of IL-15 in the serum. Functional experiments in vitro demonstrated that sustained exposure to IL-15 enhanced the expression of PD-1 and TIM-3 on both T and NK cells, indicating a causative link between high IL-15 levels and enhanced expression of TIM-3 on these cells. Receptor blockade of TIM-3 improved NK cell-mediated elimination of melanoma metastasis cell lines in vitro . These observations may lead to the development of novel biomarkers to predict patient response to checkpoint blockade treatment. They also suggest that induction of additional checkpoints is a possibility that needs to be considered when treating melanoma patients with IL-15.

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.

An important checkpoint in the progression of melanoma is the metastasis to lymph nodes. Here, to investigate the role of lymph node NK cells in disease progression, we analyze frequency, phenotype and functions of NK cells from tumour-infiltrated (TILN) and tumour-free ipsilateral lymph nodes (TFLN) of the same patients. We show an expansion of CD56(dim)CD57(dim)CD69 + CCR7 +KIR+ NK cells in TILN. TILN NK cells display robust cytotoxic activity against autologous melanoma cells. In the blood of metastatic melanoma patients, the frequency of NK cells expressing the receptors for CXCL8 receptor is increased compared with healthy subjects, and blood NK cells also express the receptors for CCL2 and IL-6. These factors are produced in high amount in TILN and in vitro switch the phenotype of blood NK cells from healthy donors to the phenotype associated with TILN. Our data suggest that the microenvironment of TILN generates and/or recruits a particularly effective NK cell subset.

Radiotherapy is the only treatment definitely indicated for diffuse pontine gliomas (DIPG). Findings on the role of EGFR signaling in the onset of childhood DIPG prompted the use of nimotuzumab, an anti-EGFR monoclonal antibody. Assuming a potential synergy with both radiotherapy and vinorelbine, a pilot phase 2 protocol was launched that combined nimotuzumab with concomitant radiation and vinorelbine. An amendment in July 2011 introduced re-irradiation at relapse. The primary endpoint for first-line treatment was objective response rate (CR + PR + SD) according to the RECIST. This report concerns the outcome of this strategy as a whole. Vinorelbine 20 mg/m(2) was administered weekly, with nimotuzumab 150 mg/m(2) in the first 12 weeks of treatment; radiotherapy was delivered from weeks 3 to 9, for a total dose of 54 Gy. Vinorelbine 25 mg/m(2) and nimotuzumab were given every other week thereafter until the tumor progressed or for up to 2 years. Re-irradiation consisted of 19.8 Gy, fractionated over 11 days. Baseline and latest MRIs were assessed blindly by an outside neuroradiologist. Twenty five children (mean age 7.4 years) were enrolled as of August 2009 (median follow-up 29 months). A response was observed in 24/25 patients (96 %). The nimotuzumab/vinorelbine combination was very well tolerated, with no acute side-effects. Eleven of 16 locally-relapsing patients were re-irradiated. One-year PFS and OS rates were 30 ± 10 % and 76 ± 9 %, respectively; 2-year OS was 27 ± 9 %; the median PFS and OS were 8.5 and 15 months, respectively. This strategy generated interesting results and warrants further investigation.

There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies., (Copyright © 2013 Elsevier Inc. All rights reserved.)