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.

Impressive response to denosumab in a patient with bone metastatic adenocarcinoma of the stomach after 2 years of zoledronic acid.

Bone is a common site for tumor spread in patients with solid tumors. So far bisphosphonates have been the main pharmacological treatment option for patients with bone metastases. We present a case of bone metastatic gastric cancer treated with zoledronic acid at first and later with denosumab. After 1 year of denosumab treatment, the 18F-fluorodeoxyglucose (18F-FDG)-PET/computed tomography scan reassessment documented a metabolic complete response, even in the absence of specific antiblastic treatment. Whether denosumab can be used directly after pretreatment with bisphosphonates has yet to be addressed.

Signal peptide-regulated toxicity of a plant ribosome-inactivating protein during cell stress.

The fate of the type I ribosome-inactivating protein (RIP) saporin when initially targeted to the endoplasmic reticulum (ER) in tobacco protoplasts has been examined. We find that saporin expression causes a marked decrease in protein synthesis, indicating that a fraction of the toxin reaches the cytosol and inactivates tobacco ribosomes. We determined that saporin is largely secreted but some is retained intracellularly, most likely in a vacuolar compartment, thus behaving very differently from the prototype RIP ricin A chain. We also find that the signal peptide can interfere with the catalytic activity of saporin when the protein fails to be targeted to the ER membrane, and that saporin toxicity undergoes signal sequence-specific regulation when the host cell is subjected to ER stress. Replacement of the saporin signal peptide with that of the ER chaperone BiP reduces saporin toxicity and makes it independent of cell stress. We propose that this stress-induced toxicity may have a role in pathogen defence.

Mutants of plant genes for developing cancer vaccines.

Preventive Human Papillomavirus (HPV) vaccination is an expensive practice and it may be an insufficient tool to tackle cervical cancer worldwide. Therapeutic intervention is seeking for safe/effective vaccines inducing the activation of CD8+ cytotoxic T lymphocytes (CTLs) that is required to clear the tumor. Linking a tumor-specific antigen (i.e. the E7 oncoprotein of the 'high risk' HPVs) to molecules able to increase its immune 'visibility' represents a strategy to force the immune system to fight cancer. We focused on plants as sources of innovative immunostimulatory sequences. We have already shown the anti-cancer activity obtained by fusing E7GGG (a mutagenized E7 gene from the high risk HPV type 16) to the coat protein of a plant virus, the Potato Virus X. We are now investigating plant-derived carriers, such as the 'Ribosome inactivating proteins' (RIPs), so far used to develop immunotoxins for targeted cancer therapy. Beside toxicity, RIPs have other features (i.e. immunogenicity, ability to modulate immune functions and apoptosis induction) that could be useful tools to use in tumor immunotherapy. A non toxic mutant of saporin (SAP-KQ) was used as a carrier for the E7GGG gene in the context of a DNA-based vaccine. We show here that fusion constructs of SAP-KQ with E7GGG can induce E7-specific Immunoglobulins (IgGs), CTLs and Delayed-Type Hypersensitivity (DTH) affecting the growth of E7-expressing tumors in mice. These data demonstrate that mutant plant genes hold promise to improve the poor immunogenicity of tumor-associated cancer antigens and could contribute to the evolution of new cancer immunotherapy.

Differential expression of saporin genes upon wounding, ABA treatment and leaf development.

Saporins are type 1 ribosome-inactivating proteins (RIPs: EC 3.2.2.22) produced in various organs of Saponaria officinalis L. Two distinct saporin types, saporin-L and saporin-S isoforms, were respectively purified from the intra- and extra-cellular fractions of soapwort leaves. The saporin-L isoform was lowly identical, differed for toxicity, molecular mass and amino acid composition from saporin-S proteins forming a new monophyletic group. Genes encoding both L- and S-type isoforms were cloned from leaf-specific cDNA library; the encoded products included the N-terminal diversity observed by protein sequencing and showed compatible weights with those from mass spectra. These genes were intron-less belonging to small gene families. Reverse transcription polymerase chain reaction/quantitative reverse transcription polymerase chain reaction experiments evidenced their differential expression during leaf development, wounding and abscisic acid treatment. These results suggest that the saporin-L and -S proteins may play diversified roles during stress responses.

Peach [Prunus persica (L.) Batsch] KNOPE1, a class 1 KNOX orthologue to Arabidopsis BREVIPEDICELLUS/KNAT1, is misexpressed during hyperplasia of leaf curl disease.

Class 1 KNOTTED-like (KNOX) transcription factors control cell meristematic identity. An investigation was carried out to determine whether they maintain this function in peach plants and might act in leaf curliness caused by the ascomycete Taphrina deformans. KNOPE1 function was assessed by overexpression in Arabidopsis and by yeast two-hybrid assays with Arabidopsis BELL proteins. Subsequently, KNOPE1 mRNA and zeatin localization was monitored during leaf curl disease. KNOPE1 and Arabidopsis BREVIPEDICELLUS (BP) proteins fell into the same phyletic group and recognized the same BELL factors. 35S:KNOPE1 Arabidopsis lines exhibited altered traits resembling those of BP-overexpressing lines. In peach shoot apical meristem, KNOPE1 was expressed in the peripheral and central zones but not in leaf primordia, identically to the BP expression pattern. These results strongly suggest that KNOPE1 must be down-regulated for leaf initiation and that it can control cell meristem identity equally as well as all class 1 KNOX genes. Leaves attacked by T. deformans share histological alterations with class 1 KNOX-overexpressing leaves, including cell proliferation and loss of cell differentiation. Both KNOPE1 and a cytokinin synthesis ISOPENTENYLTRANSFERASE gene were found to be up-regulated in infected curled leaves. At early disease stages, KNOPE1 was uniquely triggered in the palisade cells interacting with subepidermal mycelium, while zeatin vascular localization was unaltered compared with healthy leaves. Subsequently, when mycelium colonization and asci development occurred, both KNOPE1 and zeatin signals were scattered in sectors of cell disorders. These results suggest that KNOPE1 misexpression and de novo zeatin synthesis of host origin might participate in hyperplasia of leaf curl disease.

Combined vemurafenib and fotemustine in patients with BRAF V600 melanoma progressing on vemurafenib.

BACKGROUND: BRAF inhibitor vemurafenib achieves high response rate and an improvement in survival in patients with BRAF-mutated metastatic melanoma. However, median progression-free survival is only 6.9 months in the phase 3 study. Retrospective analyses suggest that treatment with BRAF inhibitors beyond initial progression might be associated with improved overall survival. We aimed to prospectively investigate the activity of prolonged treatment with vemurafenib and the addition of fotemustine in patients with systemic progression on prior single-agent BRAF inhibitor. PATIENTS AND METHODS: In this two-centres, single-arm Phase 2 trial, we enrolled patients with systemic progressive disease during single-agent vemurafenib treatment. Participants received vemurafenib 960 mg twice daily or dose administered at time of disease progression with vemurafenib previous treatment and fotemustine 100 mg/m2 intravenously every three weeks. The primary endpoint was PFS. RESULTS: Thirty-one patients were enrolled in the study; 16 patients had brain metastases at baseline. Median PFS was 3.9 months and 19 patients (61.3%) achieved disease control (1 CR, 4 PR, 14 SD). For patients achieving disease control, median duration of treatment was 6 months. Median OS was 5.8 months from enrolment and 15.4 months from start of previous vemurafenib. Five patients (16.1%) had a G3-4 AE, the most common being thrombocytopenia, which occurred in 3 patients.This trial is registered with ClinicalTrials.gov number NCT01983124. CONCLUSION: The combination of vemurafenib plus fotemustine has clinical activity and an acceptable safety profile in BRAF-refractory patients.

Current status and perspectives in immunotherapy for metastatic melanoma.

Metastatic melanoma was the first malignancy in which immune checkpoint inhibitors demonstrated their successful efficacy. Currently, the knowledge on the interaction between the immune system and malignant disease is steadily increasing and new drugs and therapeutic strategies are overlooking in the clinical scenario. To provide a comprehensive overview of immune modulating drugs currently available in the treatment of melanoma as well as to discuss of possible future strategies in the metastatic melanoma setting, the present review aims at analyzing controversial aspects about the optimal immunomodulating treatment sequences, the search for biomarkers of efficacy of immunocheckpoint inhibitors, and innovative combinations of drugs currently under investigation.

Microradiography as a tool to detect heavy metal uptake in plants for phytoremediation applications.

In this paper, an application of contact microradiography with soft X-rays for detecting the uptake site of heavy metal in the whole plant leaves is investigated. The X-ray source is a laser-plasma one based on an Nd:glass laser. The soft X-ray radiation emitted from the plasma laser targets of magnesium, iron, and copper can be strongly absorbed in the leaves' regions rich in iron, magnesium, and copper. This absorbance could point to structures in the leaves where these heavy elements are found. In this work, leaves treated with copper sulfate diluted in water at 1, 2, and 5% were imaged by using a copper target, in order to evaluate differences with untreated control leaves. Our results showed that this methodology highlighted the presence of copper in the treated leaves. This new methodology should detect heavy element pollutants inside plants and it should also be a useful analytic tool in phytoremediation studies.

Transcriptomics of tomato plants infected with TYLCSV or expressing the central TYLCSV Rep protein domain uncover changes impacting pathogen response and senescence.

To establish a successful infection viruses need to overcome plant innate immune responses and redirect host gene expression for their multiplication and diffusion. Tomato yellow leaf curl Sardinia virus (TYLCSV) is a geminivirus, which causes significant economic losses in tomato. The multifunctional replication associated geminivirus protein (Rep) has an important role during viral infection. In particular, the Rep central domain spanning from aa 120 to 180 is known to interact with viral and host factors. In this study, we used long serial analysis of gene expression to analyse the transcriptional profiles of transgenic tomato plants expressing the first 210 amino acids of TYLCSV Rep (Rep210) and TYLCSV-infected wild-type tomato plants (Wt-Ty). Also, we compared these profiles with those of transgenic Rep130 tomatoes. Comparison of Wt-Ty and Rep210 libraries with the wild-type one identified 118 and 203 differentially expressed genes (DEGs), respectively. Importantly, 55% of Wt-Ty DEGs were in common with Rep210, and no ones showed opposite expression. Conversely, a negligible overlap was found between Rep130 DEGs and Wt-Ty and Rep210 ones. TYLCSV- and Rep210-repressed genes, but not induced ones, overlapped with the leaf senescence process. Interestingly, TYLCSV upregulates expression of genes involved in the negative regulation of programmed cell death (PCD), several of which were also regulated by the abscisic acid. Rep210 upregulated genes related to defence response, immune system processes and negative regulation of PCD. Collectively, our results support a model in which the Rep central domain has a pivotal role in redirecting host plant gene expression.

Update on Metastatic Uveal Melanoma: Progress and Challenges.

Uveal melanoma is a rare and biologically distinct type of melanoma arising from melanocytes of the uveal tract; it is associated with a poor prognosis due to the lack of effective systemic treatments. Recent advances in the pathogenesis of uveal melanoma offer an unprecedented opportunity for investigation of new compounds. The purpose of this paper was to analyse the existing evidence about the molecular pathology and immunobiology of advanced uveal melanoma and their implications for systemic targeted therapies and immunotherapy, as well as to discuss future treatment strategies based on data provided by clinical and translational research studies.

Analysis of the Expression and Single-Nucleotide Variant Frequencies of the Butyrophilin-like 2 Gene in Patients With Uveal Melanoma.

IMPORTANCE: Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 (BTNL2; OMIM, 606000), which is associated with prostate cancer risk and autoimmune diseases. OBJECTIVE: To investigate the expression and variant allele frequencies of BTNL2, a candidate gene for chromosome 6 amplification, in patients with UM. DESIGN, SETTING, AND PARTICIPANTS: In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variants rs28362679 and rs41441651 with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1- and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015. MAIN OUTCOMES AND MEASURES: Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival. RESULTS: We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of the rs28362679 variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). The rs41441651 variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival. CONCLUSIONS AND RELEVANCE: Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control patients.

Expression and characterisation in E. coli of mutant forms of saporin.

In the present communication, we report on the expression and characterisation in Escherichia coli of mutant derivatives of saporin, a type 1 ribosome-inactivating protein from Saponaria officinalis L. The effects of substitution of Glu 176 with Lys and those of deletion of 19 amino acids at the C-terminal were evaluated both in vivo, testing the influence of expressed proteins on bacterial growth and in vitro measuring their N-glycosidase and supercoiled DNA relaxation activities. Results indicate that both modifications of the wild-type protein abolish its toxicity to bacterial cells and impair its enzymatic activity on polynucleotide substrates, either RNA or DNA.

Potential Role of Soluble c-Met as a New Candidate Biomarker of Metastatic Uveal Melanoma.

IMPORTANCE: Conventional melanoma serum biomarkers (S100 and lactate dehydrogenase [LDH]) perform poorly in patients with uveal melanoma, and the search for new biomarkers is needed. A high expression of the oncoprotein c-Met in primary uveal melanoma is associated with metastatic progression, and c-Met is released as a soluble ectodomain through ADAM10- and ADAM17-mediated cleavage, suggesting a possible role as biomarker. OBJECTIVE: To determine the potential role of soluble c-Met (sc-Met) as a biomarker of uveal melanoma progression in comparison with S100 and LDH. DESIGN, SETTING, AND PARTICIPANTS: Soluble c-Met was studied in the conditioned medium of 9 uveal melanoma cell lines and in the blood serum samples of 24 mice with uveal melanoma xenografts, 57 patients with uveal melanoma (17 patients whose tumors metastasized and 40 patients whose tumors did not metastasize), and 37 healthy donors. We collected blood samples for as long as 5 years after treatment of the primary tumor. The concentration of sc-Met was measured using enzyme-linked immunosorbent assays, and the receiver operating characteristic curve was used to evaluate sensitivity and specificity in the identification of metastatic uveal melanoma. The study began on May 2, 2011, and the last samples were collected in January 2015. MAIN OUTCOMES AND MEASURES: Levels of sc-Met in uveal melanoma cell cultures and in the blood serum samples of xenotransplanted mice, of healthy donors, and of patients with uveal melanoma during follow-up. RESULTS: The conditioned medium of uveal melanoma cell lines and the blood serum samples of mice with uveal melanoma xenografts contained significant levels of sc-Met. Patients with metastatic disease had significantly higher serum levels of sc-Met (median level, 590 ng/mL [range, 246-12,856 ng/mL]) than did patients without metastatic disease (median level, 296 ng/mL [range, 201-469 ng/mL]) (P < .001) and healthy donors (median level, 285 ng/mL [range, 65-463 ng/mL]) (P < .001). Analysis of receiver operating characteristic curves for sc-Met levels in patients with nonmetastatic uveal melanoma vs patients with metastatic uveal melanoma yielded an area under the curve of 0.82 (95% CI, 0.68-0.95) (P < .001), which was superior to the areas under the curve achieved with S100 or LDH markers. Patients with progressive metastatic disease showed further increases in sc-Met level, whereas stable patients did not. CONCLUSIONS AND RELEVANCE: The present pilot study suggests that sc-Met should be further exploited as a biomarker for monitoring of uveal melanoma.

Differences in X-ray absorption due to cadmium treatment in Saponaria officinalis leaves.

A method for detecting cadmium uptake in leaves of Saponaria officinalis doped with a solution of cadmium acetate is described. The technique based on the exposure of dried leaves to X-rays of a wavelength close to that of the metal K-edge could be useful for phytoremediation studies as it could reveal the bioaccumulation in plants due to the treatment either in vivo or in vitro with heavy metals. X-ray microradiography measurements are in agreement with those from peroxidase enzyme assay utilized to follow the oxidative damage induced by heavy metals. At present, as we will see in this report, microradiography has still poorer sensitivity in comparison with enzyme assay, but it has the advantage of being faster, not destructive, and usable even at very high doping levels, where the enzyme assay technique results are fully saturated. Further analysis of the optical density values could lead to a quantitative measurement of the heavy metal in the sample. Thus, the technology developed in this article could be useful for tracing the intake in phytoremediation studies.

Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (Carassius auratus).

Sexually mature goldfish (Carassius auratus) of both sexes were exposed to two doses (100 and 1000 microg/l) of the widely used herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) for a period of 21 days and effects on the concentrations of gonad and plasma sex steroids (testosterone (T), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT)), plasma vitellogenin (VTG) and gonad histo-morphology assessed. Atrazine did not show any obvious estrogenic effect in males, as determined by a lack of vitellogenin induction. There were, however, effects of atrazine on plasma androgen concentrations (androgen dynamics) and tissue (plasma and gonad) estrogen concentrations in male goldfish; exposure to 1000 microg/l atrazine induced suppression in both testosterone and 11-ketotestosterone, and resulted in elevated 17beta-estradiol, after 21 day of exposure. Further, these suppressive effects on plasma androgens and the induction in estrogen were dose- and time-related. The highest atrazine exposure dose induced structural disruption in the testis and both 100 and 1000 microg/l induced elevated levels of atresia in ovaries.

Monitoring urban air particulate matter (fractions PM 2.5 and PM 10) genotoxicity by plant systems and human cells in vitro: a comparative analysis.

Increased incidence of mortality and sickness due to cardiopulmonary complications has been associated with elevated levels of urban air particles (UAP), with an aerodynamic diameter of 10 microm (PM 10) and 2.5 microm (PM 2.5). In the present report alternative plant systems and human cells in vitro are associated with human hazard and genotoxic risk assessment of UAP. The genotoxic activities associated with the coarse (PM 10) and the fine fraction (PM 2.5) of airborne particulates have been analyzed by evaluating micronuclei induction and/or sister-chromatid exchange (SCE) using in vitro models of Daucus carota and HS 27 human fibroblast cell suspensions and Zea mays root meristems. Results show variability in the response of the test systems and indicate that the mutagenicity trend in both plant and human cell cultures was directly correlated to the concentration of carbon-rich particles in the fraction of the PM 2.5 airborne particulates. Moreover, in plant tissues, the frequency of micronuclei and SCE was related to an enhancement of the specific activity of the stress-related enzyme peroxidase.