Pilot Study to Evaluate Serum Soluble Mesothelin-Related Peptide (SMRP) as Marker for Clinical Monitoring of Pleural Mesothelioma (PM): Correlation with Modified RECIST Score.

A soluble mesothelin-related peptide (SMRP) is the only FDA-approved biomarker for diagnosis of pleural mesothelioma (PM) and the most used for monitoring treatment. Radiological assessment of PM, based on modified RECIST (mRECIST) criteria, is challenging. This pilot study was designed to evaluate whether SMRP levels correlated over time with mRECIST score. Serial serum samples from PM patients were collected and SMRP levels were measured and compared with the mRECIST score obtained through centralized CT scans by blinded review. The within-patient SMRP-mRECIST relationship over time was estimated through a normal random-effects regression approach applied to the log-transformed mRECIST score. Overall, 58 PM patients were included (46 males and 12 females) with a median age at diagnosis of 67 years (min-max = 48-79), 44 (76%) with epithelioid and 14 (24%) with non-epithelioid histology. The total number of SMRP measurements and CT scans considered for analysis was 183. There was a statistically significant correlation between SMRP and mRECIST score in the 2 cohorts considered both separately and jointly. These results, although exploratory, suggest that SMRP measurement might be considered as an adjunct to monitor PM patients in order to delay CT scans time interval, thus warranting further investigation.

Potential role of serum mesothelin in predicting survival of patients with malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor survival rates. Therefore, it is essential to have effective biological markers predicting the course of the disease and prognosis. The aim of the present study was to highlight the prognostic significance of serum soluble mesothelin-related protein (Se-SMRP) in patients with MPM at diagnosis. Se-SMRP was determined in 60 patients using an ELISA commercial kit. Se-SMRP levels were subdivided into three tertile-based categories and in each category overall survival (OS) indexes were determined using the Kaplan-Meier and Cox regression analyses. The association between Se-SMRP levels and OS was also assessed by restricted cubic spline (RCS) analysis. No notable differences in the Kaplan-Meier probabilities were identified across the Se-SMRP categories (<0.66 nM, 0.66-1.46 nM, >1.46 nM) although an upward trend in death rate ratios (RR) was pointed out by comparing the higher (RR=1.95) and intermediate (RR=1.86) categories with the lower category (RR=1.00). In addition, such an increasing tendency, particularly when the biomarker exceeded 1.0 nM, was confirmed by an RCS function of Se-SMPR levels fitted to survival data using the Cox regression equation. The present study provided evidence in favor of a prognostic value of Se-SMRP in patients with MPM.

Characterization of soluble PD-L1 in pleural effusions of mesothelioma patients: potential implications in the immune response and prognosis.

PURPOSE: Programmed death-ligand 1 (PD-L1) protein plays a central role in the antitumor immune response, and appears to be a predictor of prognosis and efficacy for PD-L1 and programmed death 1 (PD-1) blockade therapy. The immunoregulatory role and prognostic impact of PD-L1 soluble form (sPD-L1) have been investigated in biological fluids of patients with different tumors. In malignant pleural mesothelioma (MPM), circulating sPD-L1 has been recently reported in patients' sera, but no data are available in pleural effusions (PE). In our study, we evaluated the baseline expression levels of sPD-L1 in PE from 84 MPM patients and correlated them with PD-L1-status in matched tumors and patients' overall survival (OS). METHODS: sPD-L1 in PE was determined by ELISA and tumor PD-L1 by immunohistochemistry. Association of sPD-L1 with OS was estimated using the Cox regression model. RESULTS: We observed that sPD-L1 was variably expressed in all the PE and tended to be higher (by 30%) in patients with PD-L1-positive tumors (cut-off ≥ 1% stained cells) as compared to patients with PD-L1-negative tumors (geometric mean ratio = 1.28, P value = 0.288). sPD-L1 levels were significantly higher than those of sPD-1 (P value = 0.001) regardless of the MPM histotypes and they were positively correlated (r = 0.50, P value < 0.001). Moreover, high PE sPD-L1 concentrations were associated with a trend towards increased OS (hazard ratio 0.79, 95% CL 0.62-1.01, P value = 0.062). CONCLUSIONS: Our study documents the presence of sPD-L1 in PE of MPM patients, and suggests its possible biological and prognostic role in MPM.

Phenotypic characterization of tumor CTLA-4 expression in melanoma tissues and its possible role in clinical response to Ipilimumab.

The expression of the immune checkpoint molecule CTLA-4 has been almost exclusively studied in the T cell lineage, but increasing evidence has shown its expression on tumors with implications for immunotherapy. To date, the degree of expression of CTLA-4 on tumor cells as a predictive biomarker of response to immune checkpoint inhibitors has not been studied. In this report, we analyzed this issue in melanoma patients treated with CTLA-4 inhibitor Ipilimumab (IPI). We show that the level of CTLA-4 expression on melanoma cells is higher than that on tumor infiltrating lymphocytes (TIL) and it is associated with clinical response to IPI therapy supporting the idea of its possible role as a predictive biomarker.

Response to ipilimumab therapy in metastatic melanoma patients: potential relevance of CTLA-4+ tumor infiltrating lymphocytes and their in situ localization.

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3+, CD8+ T cells, and CTLA-4+ immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4+ TIL distribution in melanoma tissues taking into account localization, relationship with CD3+/CD8+ TILs, and changes in response to IPI treatment. We identified that CTLA-4+ TILs may represent a marker of IPI response, alone or with CD3+/CD8+ subsets, although this requires confirmation in larger studies.

Determination of Mesothelin Levels in Pleural Effusion Does Not Help Predict Survival of Patients With Malignant Pleural Mesothelioma.

AIM: This study evaluated the prognostic value of soluble mesothelin-related protein (SMRP) levels in pleural effusions (PE) from patients with pleural mesothelioma (MPM). PATIENTS AND METHODS: SMRP level in PE was tested using an enzyme-linked immunosorbent assay (ELISA) in 109 patients with MPM at diagnosis before any treatment. The Kaplan-Meier method and the Cox regression were applied to compare overall survival probabilities across tertile categories of SMRP level. RESULTS: No significant differences in Kaplan-Meier overall survival probabilities among the SMRP categories were found. A statistically non-significant trend for increased death rate ratio (RR) was computed (p=0.327) when the higher (>46.5 nM, RR=1.38) and intermediate (8.5-46.5 nM, RR=1.18) SMRP categories were compared to the lower category (<8.5 nM, RR=1.00). Cox regression modelling including a restricted cubic spline showed a moderately rising non-linear trend in death rate. CONCLUSION: The SMRP level in PE does not appear to have prognostic significance and its detection is not recommended in routine clinical management of patients with MPM.

Targeting of Histone Demethylases KDM5A and KDM6B Inhibits the Proliferation of Temozolomide-Resistant Glioblastoma Cells.

Lysine histone demethylases (KDMs) are considered potential therapeutic targets in several tumors, including glioblastoma (GB). In particular, KDM5A is involved in the acquisition of temozolomide (TMZ) resistance in adult GB cells and UDX/KDM6B regulates H3K27 methylation, which is involved in the pediatric diffuse intrinsic pontine glioma (DIPG). Synthetic inhibitors of KDM5A (JIB 04 and CPI-455) efficiently block the proliferation of native and TMZ-resistant cells and the KDM6B inhibitor GSK J4 improves survival in a model of DIPG. The aim of our work was to determine if GSK J4 could be effective against GB cells that have acquired TMZ resistance and if it could synergize with TMZ or JIB 04 to increase the clinical utility of these molecules. Standard functional and pharmacological analytical procedures were utilized to determine the efficacy of the molecules under study when used alone or in combination against native GB cells and in a model of drug resistance. The results of this study indicated that although GSK J4 is active against native and TMZ-resistant cells, it does so at a lower efficacy than JIB 04. Drug combination studies revealed that GSK J4, differently from JIB 04, does not synergize with TMZ. Interestingly, GSK J4 and JIB 04 strongly synergize and are a potent combination against TMZ-resistant cells. Further studies in animal models will be necessary to determine if this combination of molecules might foster the development of novel therapeutic approaches for glioblastoma.

Soluble CTLA-4 as a favorable predictive biomarker in metastatic melanoma patients treated with ipilimumab: an Italian melanoma intergroup study.

CTLA-4 blockade by means of ipilimumab (IPI) potentiates the immune response and improves overall survival (OS) in a minority of metastatic melanoma (MM) patients. We investigated the role of soluble CTLA-4 (sCTLA-4) as a possible biomarker for identifying this subset of patients. sCTLA-4 levels were analyzed at baseline in sera from 113 IPI-treated MM patients by ELISA, and the median value (200 pg/ml) was used to create two equally sized subgroups. Associations of sCTLA-4 with best overall response (BOR) to IPI and immune-related adverse events (irAEs) were evaluated through logistic regression. Kaplan-Meier and Cox regression methods were used to analyze OS. A remarkable association between sCTLA-4 levels and BOR was found. Specifically, the proportion of patients with sCTLA-4 > 200 pg/ml in irSD or irPD (immune-related stable or progressive disease) was, respectively, 80% (OR = 0.23; 95%CL = 0.03-1.88) and 89% (OR = 0.11; 95%CL = 0.02-0.71) and was lower than that observed among patients in irCR/irPR (immune-related complete/partial response). sCTLA-4 levels increased during IPI treatment, since the proportion of patients showing sCTLA > 200 pg/ml after 3 cycles was 4 times higher (OR = 4.41, 95%CL = 1.02-19.1) than that after 1 cycle. Moreover, a significantly lower death rate was estimated for patients with sCTLA-4 > 200 pg/ml (HR = 0.61, 95%CL = 0.39-0.98). Higher baseline sCTLA-4 levels were also associated with the onset of any irAE (p value = 0.029), in particular irAEs of the digestive tract (p value = 0.041). In conclusion, our results suggest that high sCTLA-4 serum levels might predict favorable clinical outcome and higher risk of irAEs in IPI-treated MM patients.

Immune Checkpoints and Innovative Therapies in Glioblastoma.

Targeting the Immune Checkpoint molecules (ICs; CTLA-4, PD-1, PD-L1/2, and others) which provide inhibitory signals to T cells, dramatically improves survival in hard-to-treat tumors. The establishment of an immunosuppressive environment prevents endogenous immune response in glioblastoma; therefore, manipulating the host immune system seems a reasonable strategy also for this tumor. In glioma patients the accumulation of CD4+/CD8+ T cells and Treg expressing high levels of CTLA-4 and PD-1, or the high expression of PD-L1 in glioma cells correlates with WHO high grade and short survival. Few clinical studies with IC inhibitors (ICis) were completed so far. Notably, the first large-scale randomized trial (NCT 02017717) that compared PD-1 blockade and anti-VEGF, did not show an OS increase in the patients treated with anti-PD-1. Several factors could have contributed to the failure of this trial and must be considered to design further clinical studies. In particular the possibility of targeting at the same time different ICs was pre-clinically tested in an animal model were inhibitors against IDO, CTLA-4 and PD-L1 were combined and showed persistent and significant antitumor effects in glioma-bearing mice. It is reasonable to hypothesize that the immunological characterization of the tumor in terms of type and level of expressed IC molecules on the tumor and TIL may be useful to design the optimal ICi combination for a given subset of tumor to overcome the immunosuppressive milieu of glioblastoma and to efficiently target a tumor with such high cellular complexity.

Epigenetic Targeting of Glioblastoma.

Glioblastoma is one of the first tumors where the biological changes accompanying a single epigenetic modification, the methylation of the MGMT gene, were found to be of clinical relevance. The exploration of the epigenomic landscape of glioblastoma has allowed to identify patients carrying a diffuse hypermethylation at gene promoters and with better outcome. Epigenetic and genetic data have led to the definition of major subgroups of glioma and were the basis of the current WHO classification of CNS tumors and of a novel classification based solely on DNA methylation data that shows a remarkable diagnostic precision.The reversibility of epigenetic modifications is considered a therapeutic opportunity in many tumors also because these alterations have been mechanistically linked to the biological characteristics of glioblastoma. Several alterations like IDH1/2 mutations that interfere with "epigenetic modifier" enzymes, the mutations of the histone 3 variants H3.1 and H3.3 that alter the global H3K27me3 levels and the altered expression of histone methyltransferases and demethylases are considered potentially druggable targets in glioma and molecules targeting these alterations are being tested in preclinical and clinical trials. The recent advances on the knowledge of the players of the "epigenetic orchestra" and of their mutual interactions are indicating new paths that may eventually open new therapeutic options for this invariably lethal cancer.

Corrigendum: Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

[This corrects the article on p. 386 in vol. 8, PMID: 28446908.].

MicroRNA in Glioblastoma: An Overview.

Glioblastoma is the most aggressive brain tumor and, even with the current multimodal therapy, is an invariably lethal cancer with a life expectancy that depends on the tumor subtype but, even in the most favorable cases, rarely exceeds 2 years. Epigenetic factors play an important role in gliomagenesis, are strong predictors of outcome, and are important determinants for the resistance to radio- and chemotherapy. The latest addition to the epigenetic machinery is the noncoding RNA (ncRNA), that is, RNA molecules that are not translated into a protein and that exert their function by base pairing with other nucleic acids in a reversible and nonmutational mode. MicroRNAs (miRNA) are a class of ncRNA of about 22 bp that regulate gene expression by binding to complementary sequences in the mRNA and silence its translation into proteins. MicroRNAs reversibly regulate transcription through nonmutational mechanisms; accordingly, they can be considered as epigenetic effectors. In this review, we will discuss the role of miRNA in glioma focusing on their role in drug resistance and on their potential applications in the therapy of this tumor.

Small molecules targeting histone demethylase genes (KDMs) inhibit growth of temozolomide-resistant glioblastoma cells.

In glioblastoma several histone demethylase genes (KDM) are overexpressed compared to normal brain tissue and the development of Temozolomide (TMZ) resistance is accompanied by the transient further increased expression of KDM5A and other KDMs following a mechanism that we defined as "epigenetic resilience". We hypothesized that targeting KDMs may kill the cells that survive the cytotoxic therapy.We determined the effect of JIB 04 and CPI-455, two KDM inhibitors, on glioblastoma cells and found that both molecules are more effective against TMZ-resistant rather than native cells.Because of its lower IC50, we focused on JIB 04 that targets KDM5A and other KDMs as well. We have shown that this molecule activates autophagic and apoptotic pathways, interferes with cell cycle progression, inhibits cell clonogenicity and dephosphorylates Akt thus inactivating a potent pro-survival pathway. We performed combination temozolomide/JIB 04 in vitro treatments showing that these two molecules, under certain conditions, have a strong synergic effect and we hypothesize that JIB 04 intercepts the cells that escape the G2 block exerted by TMZ. Finally we studied the permeability of JIB 04 across the blood-brain barrier and found that this molecule reaches bioactive concentration in the brain; furthermore a pilot in vivo experiment in an orthotopic GB xenograft model showed a trend toward longer survival in treated mice with an Hazard Ratio of 0.5.In conclusion we propose that the combination between cytotoxic drugs and molecules acting on the epigenetic landscape may offer the opportunity to develop new therapies for this invariably lethal disease.

Association of CTLA-4 Gene Variants with Response to Therapy and Long-term Survival in Metastatic Melanoma Patients Treated with Ipilimumab: An Italian Melanoma Intergroup Study.

Ipilimumab (IPI) blocks CTLA-4 immune checkpoint resulting in T cell activation and enhanced antitumor immunity. IPI improves overall survival (OS) in 22% of patients with metastatic melanoma (MM). We investigated the association of CTLA-4 single nucleotide variants (SNVs) with best overall response (BOR) to IPI and OS in a cohort of 173 MM patients. Patients were genotyped for six CTLA-4 SNVs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, and CT60G>A). We assessed the association between SNVs and BOR through multinomial logistic regression (MLR) and the prognostic effect of SNVs on OS through Kaplan-Meier method. Both -1577G>A and CT60G>A SNVs were found significantly associated with BOR. In particular, the proportion of responders was higher in G/G genotype while that of stable patients was higher in A/A genotype. The frequency of patients experiencing progression was similar in all genotypes. MLR evidenced a strong downward trend in the probability of responsiveness/progression, in comparison to disease stability, as a function of the allele A "dose" (0, 1, or 2) in both SNVs with reductions of about 70% (G/A vs G/G) and about 95% (A/A vs G/G). Moreover, -1577G/G and CT60G/G genotypes were associated with long-term OS, the surviving patients being at 3 years 29.8 and 30.8%, respectively, as compared to 12.9 and 14.4% of surviving patients carrying -1577G/A and CT60G/A, respectively. MM patients carrying -1577G/G or CT60G/G genotypes may benefit from IPI treatment in terms of BOR and long-term OS. These CTLA-4 SNVs may serve as potential biomarkers predictive of favorable outcome in this subset of patients.