Case Report: Circulating Myeloid-Derived Suppressive-Like Cells and Exhausted Immune Cells in Non-Small Cell Lung Cancer Patients Treated With Three Immune Checkpoint Inhibitors.

Immune checkpoint inhibition induced a great step forward in the treatment of non-small cell lung cancer patients. In cancer immune microenvironment many checkpoints were studied and their involvement could represent a mechanism of resistance to cancer immunotherapy. For this reason, the inhibition of multiple immune checkpoints is under development. However, myeloid-derived suppressor cells (MDSC) and exhausted immune cells could limit the efficacy of cancer immunotherapy. We analyzed the variation of circulating immune suppressive-like cell subsets and exhausted immune cells in three non-small cell lung cancer patients treated with the combination of anti-CTLA-4 plus anti-PD-1 plus anti-LAG-3 at T0 (baseline), T1 (after 2 months) and T2 (after 4 months). We also describe the clinical and radiological course of the disease during this treatment in all three patients. We observed both clinical differences and changes in the composition of immune suppressive-like cell subsets and exhausted immune cells between the patients receiving the same schedule of treatment with immune checkpoint inhibitors. The study on a wider patient population and experimental model design could help to clarify the kinetics of these cell subpopulations with the perspective to find new targets for treatment or new biomarkers for resistance to cancer immunotherapy.

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water.

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.

Glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-I xenobiotic metabolizing enzymes and increases free radical generation in rat liver.

Epidemiological and animal studies linking high fruit and vegetable consumption to lower cancer risk have strengthened the belief that long-term administration of isolated naturally occurring dietary constituents could reduce the risk of cancer. In recent years, metabolites derived from phytoalexins, such as glucoraphanin found in broccoli and other cruciferous vegetables (Brassicaceae), have gained much attention as potential cancer chemopreventive agents. The protective effect of these micronutrients is assumed to be due to the inhibition of Phase-I carcinogen-bioactivating enzymes and/or induction of Phase-II detoxifying enzymes, an assumption that still remains uncertain. The protective effect of glucoraphanin is thought to be due to sulforaphane, an isothiocyanate metabolite produced from glucoraphanin by myrosinase. Here we show, in rat liver, that while glucoraphanin slightly induces Phase-II enzymes, it powerfully boosts Phase-I enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), nitrosamines and olefins. Induction of the cytochrome P450 (CYP) isoforms CYP1A1/2, CYP3A1/2 and CYP2E1 was confirmed by Western immunoblotting. CYP induction was paralleled by an increase in the corresponding mRNA levels. Concomitant with this Phase-I induction, we also found that glucoraphanin generated large amount of various reactive radical species, as determined by electron paramagnetic resonance (EPR) spectrometry coupled to a radical-probe technique. This suggests that long-term uncontrolled administration of glucoraphanin could actually pose a potential health hazard.

Aromatase inhibitors and anti-synthetase syndrome.

Adjuvant therapy in postmenopausal women with endocrine-responsive breast cancer (BC) is actually centered on the use of anti-aromatase inhibitors (AI). Several reports, however, are emerging in literature associating the use of this drugs to rheumatic disorders. This case report describes the first case of anti-synthetase syndrome diagnosis after treatment with anti-estrogen agents in a patient with pre-existing rheumatoid arthritis.

Gender differences in genetic damage induced by the tobacco-specific nitrosamine NNK and the influence of the Thr241Met polymorphism in the XRCC3 gene.

The rapid increase in adenocarcinoma of the lung and mortality amongst women strongly suggests that gender differences exist in sensitivity to certain tobacco carcinogens. In the current study, we performed the mutagen-sensitivity assay, with the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), to test the hypothesis that women are more sensitive to the genotoxic effects of NNK than men. Chromosome aberration (CA) frequencies in peripheral blood lymphocytes (PBLs) from 99 patients were evaluated before and after in vitro exposure to NNK. Because the Thr241Met polymorphism in the DNA-repair gene XRCC3 is associated with increased risk of tobacco-related cancers, especially among women, we also tested the hypothesis that individuals who inherit the homozygous variant 241Met allele are more sensitive to the genotoxic effects of NNK. CA frequency was significantly higher 1 hr after NNK treatment in women, compared with men (P = 0.02). When smoking and gender were considered together, a significant interaction was observed. PBLs from female smokers had significantly higher frequencies of NNK-induced CA, compared with female nonsmokers 1 hr after treatment (P = 0.02). We observed no overall effect of the Thr241Met polymorphism on NNK-induced CA in men, women, smokers, or nonsmokers. Overall, our data indicate that women are more sensitive to the genotoxic effects of NNK than men. Because in past years smoking among women has increased, and in view of the close correlation between NNK exposure and adenocarcinoma of the lung, our data provide a plausible explanation for the recent increase in the incidence of this cancer among women.

In vitro induction of benzo(a)pyrene cell-transforming activity by the glucosinolate gluconasturtiin found in cruciferous vegetables.

Cytotoxic and cell-transforming activity of gluconasturtiin (GNST), a promising chemopreventive agent commonly found in human diet, was studied in a medium-term bioassay utilizing BALB/c 3T3 cells. We also assessed whether GNST coupled with myrosinase, thus yielding product phenylethyl isothiocyanate (as shown by gas chromatography-mass spectral analysis), can affect the transforming potential of benzo(a)pyrene (B(a)P). Neither cytotoxicity nor cell-transforming activity was recorded. On the contrary, a marked increase (up to sevenfold) of the transforming activity of B(a)P was seen. This cocarcinogenic potential could be ascribed to an imbalance among bioactivation/detoxication during cell growth. These results indicate the need for an overall toxicological characterization of a chemopreventive agent prior to large-scale use.

Effect of XPD/ERCC2 polymorphisms on chromosome aberration frequencies in smokers and on sensitivity to the mutagenic tobacco-specific nitrosamine NNK.

Polymorphisms in DNA-repair genes could contribute to the interindividual differences in cancer susceptibility in smokers. By reducing DNA-repair capacity, these polymorphisms may influence the net level of smoking-induced genetic damage significantly, a critical step in the cascade of events leading to cancer. In this biomonitoring study, we examined the relationship between polymorphisms in the DNA-repair gene XPD/ERCC2 and genetic damage. We tested the hypothesis that coding polymorphisms in XPD/ERCC2 limit DNA-repair efficiency in humans leading to increased frequencies of chromosome aberration (CA) in their lymphocytes. We also used the mutagen-sensitivity assay, with the tobacco-specific nitrosamine NNK as a model mutagen, to determine whether lymphocytes from individuals with the variant XPD alleles are more sensitive to this tobacco-specific carcinogen. We calculated odds ratios (ORs) as estimates of relative risk of increased frequencies of CA associated with two XPD polymorphisms (Asp312Asn in exon 10 and Lys751Gln in exon 23). We observed a 2.57-fold (95% confidence limit [CL] = 0.88-7.50; P = 0.10) increase in risk of elevated in vivo frequencies of CA associated with the variant 312Asn allele in the total population. The relative risk was more pronounced in smokers (OR = 4.67; 95% CL = 1.04-20.90; P = 0.04) and in all subjects >48 years old (OR = 7.33; 95% CL = 1.53-35.10; P = 0.01). Similarly, elevations in NNK-induced aberrations were significantly associated with the 312Asn allele (OR = 3.69; 95% CL = 1.29-10.56; P = 0.02). The risk was higher in smokers (OR = 4.62; 95% CL = 1.14-18.70; P = 0.04) and in subjects >48 years old (OR = 5.76; 95% CL = 1.30-25.41; P = 0.03). No significant effect was observed with the 715Gln variant allele in relation to either in vivo or NNK-induced CA. These data suggest that the Asp312Asn polymorphism may alter the phenotype of the XPD protein, resulting in reduced DNA-repair capacity.

Induction and suppression of cytochrome P450 isoenzymes and generation of oxygen radicals by procymidone in liver, kidney and lung of CD1 mice.

Although chronic administration of procymidone (a widely used dicarboximide fungicide) leads to an increased incidence of liver tumors in mice, short-term genotoxicity studies proved negative. As cytochrome P450 (CYP) induction has been linked to non-genotoxic carcinogenesis, we investigated whether procymidone administration causes induction of CYP-dependent monooxygenases in liver, kidney and lung microsomes of male Swiss Albino CD1 mice after single or repeated (daily for three consecutive days) i.p. treatment with either 400 or 800 (1/10 or 1/20 of the DL(50)) mgkg(-1) b.w. procymidone. CYP content and CYP3A1/2, 1A1, 1A2, 2B1/2, 2E1, 2A, 2D9 and 2C11 supported oxidations were studied using either the regio- and stereo-selective hydroxylation of testosterone as multibiomarker or highly specific substrates as probes of various CYPs. While a single dose was uneffective, multiple procymidone administration lead to marked inductions of various monooxygenases: CYP3A1/2 in liver and lung (as measured by N-demethylation of aminopyrine and testosterone 6 beta-hydroxylase); CYP2E1 in liver (p-nitrophenol hydroxylation); CYP1A1 in liver and kidney (deethylation of ethoxyresorufin). Several hydroxylations were induced in the liver, including the CYP2A-linked 7 alpha (14-fold) as well as 6 alpha (22-fold), 6 beta, 16 beta and 2 beta hydroxylases. The pattern of inductions/suppressions recorded in the three different tissues suggests that procymidone exerts complex effects on the CYP profile. Tissue-specific trends included a large number of inductions in the liver and suppressions in the lung. The main inductions were corroborated by immunoblotting analyses and Northern blotting showed that inductions of CYP3A1/2, CYP2E1 and CYP1A1/2 were paralleled by increased mRNA levels. It was also found that CYP over-expression generates large amounts of reactive oxygen species (ROS), especially in liver. These data may explain why in vitro short-term genotoxicity studies on procymidone were negative, whereas in vivo long-term carcinogenesis studies turned out positive: long-term CYP induction (e.g. oxygen centered free radicals over-production) can have a co-carcinogenic and/or promoting potential.

Common commercial and consumer products contain activators of the aryl hydrocarbon (dioxin) receptor.

Activation of the Ah receptor (AhR) by halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), can produce a wide variety of toxic and biological effects. While recent studies have shown that the AhR can bind and be activated by structurally diverse chemicals, how widespread of these AhR agonists are in environmental, biological and synthetic materials remains to be determined. Using AhR-based assays, we demonstrate the presence of potent AhR agonists in a variety of common commercial and consumer items. Solvent extracts of paper, rubber and plastic products contain chemicals that can bind to and stimulate AhR DNA binding and/or AhR-dependent gene expression in hepatic cytosol, cultured cell lines, human epidermis and zebrafish embryos. In contrast to TCDD and other persistent dioxin-like HAHs, activation of AhR-dependent gene expression by these extracts was transient, suggesting that the agonists are metabolically labile. Solvent extracts of rubber products produce AhR-dependent developmental toxicity in zebrafish in vivo, and inhibition of expression of the metabolic enzyme CYP1A, significantly increased their toxic potency. Although the identity of the responsible AhR-active chemicals and their toxicological impact remain to be determined, our data demonstrate that AhR active chemicals are widely distributed in everyday products.

Lipoplatin treatment in lung and breast cancer.

The introduction of cisplatin in cancer treatment represents an important achievement in the oncologic field. Many types of cancers are now treated with this drug, and in testicular cancer patients major results are reached. Since 1965, other compounds were disovered and among them carboplatin and oxaliplatin are the main Cisplatin analogues showing similar clinical efficacy with a safer toxicity profile. Lipoplatin is a new liposomal cisplatin formulation which seems to have these characteristics. Lipoplatin was shown to be effective in NSCLC both in phase 2 and phase 3 trials, with the same response rate of Cisplatin, a comparable overall survival but less toxicity. A new protocol aiming to elucidate the double capacity of Lipoplatin to act as a chemotherapeutic and angiogenetic agent in triple-negative breast cancer patients is upcoming.

The novel radical scavenger IAC is effective in preventing and protecting against post-ischemic brain damage in Mongolian gerbils.

The removal of pathologically generated free radicals produced during ischemia, reperfusion and intracranical hemorrhage seems to be a viable approach to neuroprotection. However, at present, no neuroprotective agent has proven effective in focal ischemic stroke phase III trials, despite the encouraging data in animal models. This study aimed to explore the effect of the brain penetrant low molecular weight radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC) in neurological damage subsequent to ischemia-reperfusion injury in Mongolian gerbils. We examined the intraperitoneal effects of IAC on temporary bilateral common carotid artery occlusion (BCCO) by means of morphological and histological analysis of the hippocampus. Significant dose-dependent protective effects of IAC (1 to 10mg/kg b.w.) against neuropathological and morphological brain changes were seen when administered i.p. 1h before temporary BCCO in Mongolian gerbils. When administered up to 6h after BCCO, IAC actually reverses the neurodegenerative processes (e.g. hippocampal cell viability) induced by ischemia in a dose-dependent fashion. Data show that IAC is highly effective in protecting and preventing oxidative brain damage associated with cerebral flow disturbances. It is also effective even in late treatment of the insult, emphasizing its potential role for the management of ischemic stroke patients.