SNP of Aromatase Predict Long-term Survival and Aromatase Inhibitor Toxicity in Patients with Early Breast Cancer: A Biomarker Analysis of the GIM4 and GIM5 Trials.

PURPOSE: In estrogen receptor-positive (ER+) breast cancer, single-nucleotide polymorphisms (SNP) in the aromatase gene might affect aromatase inhibitors (AI) metabolism and efficacy. Here, we assessed the impact of SNP on prognosis and toxicity of patients receiving adjuvant letrozole. EXPERIMENTAL DESIGN: We enrolled 886 postmenopausal patients in the study. They were treated with letrozole for 2 to 5 years after taking tamoxifen for 2 to 6 years, continuing until they completed 5 to 10 years of therapy. Germline DNA was genotyped for SNP rs4646, rs10046, rs749292, and rs727479. Log-rank test and Cox model were used for disease-free survival (DFS) and overall survival (OS). Cumulative incidence (CI) of breast cancer metastasis was assessed through competing risk analysis, with contralateral breast cancer, second malignancies and non-breast cancer death as competing events. CI of skeletal and cardiovascular events were assessed using DFS events as competing events. Subdistribution HR (sHR) with 95% confidence intervals were calculated through Fine-Gray method. RESULTS: No SNP was associated with DFS. Variants rs10046 [sHR 2.03, (1.04-2.94)], rs749292 [sHR 2.11, (1.12-3.94)], and rs727479 [sHR 2.62, (1.17-5.83)] were associated with breast cancer metastasis. Three groups were identified on the basis of the number of these variants (0, 1, >1). Variant-based groups were associated with breast cancer metastasis (10-year CI 2.5%, 7.6%, 10.7%, P = 0.035) and OS (10-year estimates 96.5%, 93.0%, 89.6%, P = 0.030). Co-occurrence of rs10046 and rs749292 was negatively associated with 10-year CI of skeletal events (3.2% vs. 10%, P = 0.033). A similar association emerged between rs727479 and cardiovascular events (0.3% vs. 2.1%, P = 0.026). CONCLUSIONS: SNP of aromatase gene predict risk of metastasis and AI-related toxicity in ER+ early breast cancer, opening an opportunity for better treatment individualization.

Circulating miRNAs in Breast Cancer Diagnosis and Prognosis.

Great improvement has been made in the diagnosis and therapy of breast cancer patients. However, the identification of biomarkers for early diagnosis, prognosis, therapy assessment and monitoring, including drug resistance and the early detection of micro-metastases, is still lacking. Recently, circulating microRNAs (miRNAs), circulating freely in the blood stream or entrapped in extracellular vesicles (EVs), have been shown to have a potential diagnostic, prognostic or predictive power. In this review, recent findings are summarized, both at a preclinical and clinical level, related to miRNA applicability in the context of breast cancer. Different aspects, including clinical and technical challenges, are discussed, describing the potentialities of miRNA use in breast cancer. Even though more methodological standardized studies conducted in larger and selected patient cohorts are needed to support the effective clinical utility of miRNA as biomarkers, they could represent novel and accessible tools to be transferred into clinical practice.

Therapeutic efficacy of proton transport inhibitors alone or in combination with cisplatin in triple negative and hormone sensitive breast cancer models.

Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+  T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates.

Two high-rate pentose-phosphate pathways in cancer cells.

The relevant role of pentose phosphate pathway (PPP) in cancer metabolic reprogramming has been usually outlined by studying glucose-6-phosphate dehydrogenase (G6PD). However, recent evidence suggests an unexpected role for a less characterized PPP, triggered by hexose-6-phosphate dehydrogenase (H6PD) within the endoplasmic reticulum (ER). Studying H6PD biological role in breast and lung cancer, here we show that gene silencing of this reticular enzyme decreases cell content of PPP intermediates and D-ribose, to a similar extent as G6PD silencing. Decrease in overall NADPH content and increase in cell oxidative status are also comparable. Finally, either gene silencing impairs at a similar degree cell proliferating activity. This unexpected response occurs despite the absence of any cross-interference between the expression of both G6PD and H6PD. Thus, overall cancer PPP reflects the contribution of two different pathways located in the cytosol and ER, respectively. Disregarding the reticular pathway might hamper our comprehension of PPP role in cancer cell biology.

Mechanisms underlying the predictive power of high skeletal muscle uptake of FDG in amyotrophic lateral sclerosis.

BACKGROUND: We recently reported that enhanced [18F]-fluorodeoxyglucose (FDG) uptake in skeletal muscles predicts disease aggressiveness in patients with amyotrophic lateral sclerosis (ALS). The present experimental study aimed to assess whether this predictive potential reflects the link between FDG uptake and redox stress that has been previously reported in different tissues and disease models. METHODS: The study included 15 SOD1G93A mice (as experimental ALS model) and 15 wildtype mice (around 120 days old). Mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested quadriceps and hearts by biochemical, immunohistochemical, and immunofluorescence analysis. Colocalization between the endoplasmic reticulum (ER) and the fluorescent FDG analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was performed in fresh skeletal muscle sections. Finally, mitochondrial ultrastructure and bioenergetics were evaluated in harvested quadriceps and hearts. RESULTS: FDG retention was significantly higher in hindlimb skeletal muscles of symptomatic SOD1G93A mice with respect to control ones. This difference was not explained by any acceleration in glucose degradation through glycolysis or cytosolic pentose phosphate pathway (PPP). Similarly, it was independent of inflammatory infiltration. Rather, the high FDG retention in SOD1G93A skeletal muscle was associated with an accelerated generation of reactive oxygen species. This redox stress selectively involved the ER and the local PPP triggered by hexose-6P-dehydrogenase. ER involvement was confirmed by the colocalization of the 2-NBDG with a vital ER tracker. The oxidative damage in transgenic skeletal muscle was associated with a severe impairment in the crosstalk between ER and mitochondria combined with alterations in mitochondrial ultrastructure and fusion/fission balance. The expected respiratory damage was confirmed by a deceleration in ATP synthesis and oxygen consumption rate. These same abnormalities were represented to a markedly lower degree in the myocardium, as a sample of non-voluntary striated muscle. CONCLUSION: Skeletal muscle of SOD1G93A mice reproduces the increased FDG uptake observed in ALS patients. This finding reflects the selective activation of the ER-PPP in response to significant redox stress associated with alterations of mitochondrial ultrastructure, networking, and connection with the ER itself. This scenario is less severe in cardiomyocytes suggesting a relevant role for either communication with synaptic plaque or contraction dynamics.

The Elusive Link Between Cancer FDG Uptake and Glycolytic Flux Explains the Preserved Diagnostic Accuracy of PET/CT in Diabetes.

This study aims to verify in experimental models of hyperglycemia induced by streptozotocin (STZ-DM) to what degree the high competition between unlabeled glucose and metformin (MET) treatment might affect the accuracy of cancer FDG imaging. The study included 36 "control" and 36 "STZ-DM" Balb/c mice, undergoing intraperitoneal injection of saline or streptozotocin, respectively. Two-weeks later, mice were subcutaneously implanted with breast (4 T1) or colon (CT26) cancer cells and subdivided in three subgroups for treatment with water or with MET at 10 or 750 mg/Kg/day. Two weeks after, mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested tumors. Finally, competition by glucose, 2-deoxyglucose (2DG) and the fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) on FDG uptake was studied in 4 T1 and CT26 cultured cells. STZ-DM slightly decreased cancer volume and FDG uptake rate (MRF). More importantly, it also abolished MET capability to decelerate lesion growth and MRF. This metabolic reprogramming closely agreed with the activity of hexose-6-phosphate dehydrogenase within the endoplasmic reticulum. Finally, co-incubation with 2DG virtually abolished FDG and 2-NBDG uptake within the endoplasmic reticulum in cultured cells. These data challenge the current dogma linking FDG uptake to glycolytic flux and introduce a new model to explain the relation between glucose analogue uptake and hexoses reticular metabolism. This selective fate of FDG contributes to the preserved sensitivity of PET imaging in oncology even in chronic moderate hyperglycemic conditions.

FDG uptake tracks the oxidative damage in diabetic skeletal muscle: An experimental study.

OBJECTIVES: The present study aims to verify the relationship between glucose consumption and uptake of 18F-2-deoxy-glucose (FDG) in the skeletal muscle (SM) of experimental models of streptozotocin-induced diabetes mellitus (STZ-DM). METHODS: The study included 36 Balb/c mice. Two weeks after intraperitoneal administration of saline (control group, n = 18) or 150 mg streptozotocin (STZ-DM group, n = 18), the two cohorts were submitted to an oral glucose tolerance test and were further subdivided into three groups (n = 6 each): untreated and treated with metformin (MTF) at low or high doses (10 or 750 mg/kg daily, respectively). Two weeks thereafter, all mice were submitted to dynamic micro-positron emission tomography (PET) imaging after prolonged fasting. After sacrifice, enzymatic pathways and response to oxidative stress were evaluated in harvested SM. RESULTS: On PET imaging, the FDG uptake rate in hindlimb SM was significantly lower in nondiabetic mice as compared with STZ-DM-untreated mice. MTF had no significant effect on SM FDG uptake in untreated mice; however, its high dose induced a significant decrease in STZ-DM animals. Upon conventional analysis, the SM standard uptake value was higher in STZ-DM mice, while MTF was virtually ineffective in either control or STZ-DM models. This metabolic reprogramming was not explained by any change in cytosolic glucose metabolism. By contrast, it closely agreed with the catalytic function of hexose-6P-dehydrogenase (H6PD; i.e., the trigger of a specific pentose phosphate pathway selectively located within the endoplasmic reticulum). In agreement with this role, the H6PD enzymatic response to both STZ-DM and MTF matched the activation of the NADPH-dependent antioxidant responses to the increased generation of reactive oxygen species caused by chronic hyperglycemia. Ex vivo analysis of tracer kinetics confirmed that the enhanced SM avidity for FDG occurred despite a significant reduction in glucose consumption, while it was associated with increased radioactivity transfer to the endoplasmic reticulum. CONCLUSIONS: These data challenge the current dogma linking FDG uptake to the glycolytic rate. They instead introduce a new model considering a strict link between the uptake of this glucose analog, H6PD reticular activity, and oxidative damage in diabetes, at least under fasting condition.

Increased myocardial 18F-FDG uptake as a marker of Doxorubicin-induced oxidative stress.

BACKGROUND: Oxidative stress and its interference on myocardial metabolism play a major role in Doxorubicin (DXR) cardiotoxic cascade. METHODS: Mice models of neuroblastoma (NB) were treated with 5 mg DXR/kg, either free (Free-DXR) or encapsulated in untargeted (SL[DXR]) or in NB-targeting Stealth Liposomes (pep-SL[DXR] and TP-pep-SL[DXR]). Control mice received saline. FDG-PET was performed at baseline (PET1) and 7 days after therapy (PET2). At PET2 Troponin-I and NT-proBNP were assessed. Explanted hearts underwent biochemical, histological, and immunohistochemical analyses. Finally, FDG uptake and glucose consumption were simultaneously measured in cultured H9c2 in the presence/absence of Free-DXR (1 µM). RESULTS: Free-DXR significantly enhanced the myocardial oxidative stress. Myocardial-SUV remained relatively stable in controls and mice treated with liposomal formulations, while it significantly increased at PET2 with respect to baseline in Free-DXR. At this timepoint, myocardial-SUV was directly correlated with both myocardial redox stress and hexose-6-phosphate-dehydrogenase (H6PD) enzymatic activity, which selectively sustain cellular anti-oxidant mechanisms. Intriguingly, in vitro, Free-DXR selectively increased FDG extraction fraction without altering the corresponding value for glucose. CONCLUSION: The direct correlation between cardiac FDG uptake and oxidative stress indexes supports the potential role of FDG-PET as an early biomarker of DXR oxidative damage.

G6Pase location in the endoplasmic reticulum: Implications on compartmental analysis of FDG uptake in cancer cells.

The favourable kinetics of 18F-fluoro-2-deoxyglucose (FDG) permits to depict cancer glucose consumption by a single evaluation of late tracer uptake. This standard procedure relies on the slow radioactivity loss, usually attributed to the limited tumour expression of G6P-phosphatase (G6Pase). However, this classical interpretation intrinsically represents an approximation since, as in all tissues, cancer G6Pase activity is remarkable and is confined to the endoplasmic reticulum (ER), whose lumen must be reached by phosphorylated FDG to explain its hydrolysis and radioactivity release. The present study tested the impact of G6Pase sequestration on the mathematical description of FDG trafficking and handling in cultured cancer cells. Our data show that accounting for tracer access to the ER configures this compartment as the preferential site of FDG accumulation. This is confirmed by the reticular localization of fluorescent FDG analogues. Remarkably enough, reticular accumulation rate of FDG is dependent upon extracellular glucose availability, thus configuring the same ER as a significant determinant of cancer glucose metabolism.

Obligatory role of endoplasmic reticulum in brain FDG uptake.

PURPOSE: The endoplasmic reticulum (ER) contains hexose-6P-dehydrogenase (H6PD). This enzyme competes with glucose-6P-phosphatase for processing a variety of phosphorylated hexoses including 2DG-6P. The present study aimed to verify whether this ER glucose-processing machinery contributes to brain FDG uptake. METHODS: Effect of the H6PD inhibitor metformin on brain 18F-FDG accumulation was studied, in vivo, by microPET imaging. These data were complemented with the in vitro estimation of the lumped constant (LC). Finally, reticular accumulation of the fluorescent 2DG analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2NBDG) and its response to metformin was studied by confocal microscopy in cultured neurons and astrocytes. RESULTS: Metformin halved brain 18F-FDG accumulation without altering whole body tracer clearance. Ex vivo, this same response faced the doubling of both glucose consumption and lactate release. The consequent fall in LC was not explained by any change in expression or activity of its theoretical determinants (GLUTs, hexokinases, glucose-6P-phosphatase), while it agreed with the drug-induced inhibition of H6PD function. In vitro, 2NBDG accumulation selectively involved the ER lumen and correlated with H6PD activity being higher in neurons than in astrocytes, despite a lower glucose consumption. CONCLUSIONS: The activity of the reticular enzyme H6PD profoundly contributes to brain 18F-FDG uptake. These data challenge the current dogma linking 2DG/FDG uptake to the glycolytic rate and introduce a new model to explain the link between 18-FDG uptake and neuronal activity.

Effect of starvation on brain glucose metabolism and 18F-2-fluoro-2-deoxyglucose uptake: an experimental in-vivo and ex-vivo study.

BACKGROUND: The close connection between neuronal activity and glucose consumption accounts for the clinical value of 18F-fluoro-2-deoxyglucose (FDG) imaging in neurodegenerative disorders. Nevertheless, brain metabolic response to starvation (STS) might hamper the diagnostic accuracy of FDG PET/CT when the cognitive impairment results in a severe food deprivation. METHODS: Thirty six-week-old BALB/c female mice were divided into two groups: "control" group (n = 15) were kept under standard conditions and exposed to fasting for 6 h before the study; the remaining "STS" mice were submitted to 48 h STS (absence of food and free access to water) before imaging. In each group, nine mice were submitted to dynamic micro-PET imaging to estimate brain and skeletal muscle glucose consumption (C- and SM-MRGlu*) by Patlak approach, while six mice were sacrificed for ex vivo determination of the lumped constant, defined as the ratio between CMRGlu* and glucose consumption measured by glucose removal from the incubation medium (n = 3) or biochemical analyses (n = 3), respectively. RESULTS: CMRGlu* was lower in starved than in control mice (46.1 ± 23.3 vs 119.5 ± 40.2 nmol × min-1 × g-1, respectively, p < 0.001). Ex vivo evaluation documented a remarkable stability of lumped constant as documented by the stability of GLUT expression, G6Pase activity, and kinetic features of hexokinase-catalyzed phosphorylation. However, brain SUV in STS mice was even (though not significantly) higher with respect to control mice. Conversely, a marked decrease in both SM-MRGlu* and SM-SUV was documented in STS mice with respect to controls. CONCLUSIONS: STS markedly decreases brain glucose consumption without altering measured FDG SUV in mouse experimental models. This apparent paradox does not reflect any change in lumped constant. Rather, it might be explained by the metabolic response of the whole body: the decrease in FDG sequestration by the skeletal muscle is as profound as to prolong tracer persistence in the bloodstream and thus its availability for brain uptake.

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.].

Restoring microenvironmental redox and pH homeostasis inhibits neoplastic cell growth and migration: therapeutic efficacy of esomeprazole plus sulfasalazine on 3-MCA-induced sarcoma.

Neoplastic cells live in a stressful context and survive thanks to their ability to overcome stress. Thus, tumor cell responses to stress are potential therapeutic targets. We selected two such responses in melanoma and sarcoma cells: the xc- antioxidant system, that opposes oxidative stress, and surface v-ATPases that counteract the low pHi by extruding protons, and targeted them with the xc- blocker sulfasalazine and the proton pump inhibitor esomeprazole. Sulfasalazine inhibited the cystine/cysteine redox cycle and esomeprazole decreased pHi while increasing pHe in tumor cell lines. Although the single treatment with either drug slightly inhibited cell proliferation and motility, the association of sulfasalazine and esomeprazole powerfully decreased sarcoma and melanoma growth and migration. In the 3-methylcholanthrene (3-MCA)-induced sarcoma model, the combined therapy strongly reduced the tumor burden and increased the survival time: notably, 22 % of double-treated mice recovered and survived off therapy. Tumor-associated macrophages (TAM) displaying M2 markers, that abundantly infiltrate sarcoma and melanoma, overexpress xc- and membrane v-ATPases and were drastically decreased in tumors from mice undergone the combined therapy. Thus, the double targeting of tumor cells and macrophages by sulfasalazine and esomeprazole has a double therapeutic effect, as decreasing TAM infiltration deprives tumor cells of a crucial allied. Sulfasalazine and esomeprazole may therefore display unexpected therapeutic values, especially in case of hard-to-treat cancers.

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.

Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines.

BACKGROUND: Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. PATIENTS AND METHODS: Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients' peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by (51)Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. RESULTS: We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). CONCLUSIONS: Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory.

A novel multiplex pyrosequencing assay for genotyping functionally relevant CTLA-4 polymorphisms: potential applications in autoimmunity and cancer.

CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT diplotype and -319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto's thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects.

TLR costimulation causes oxidative stress with unbalance of proinflammatory and anti-inflammatory cytokine production.

IL-1ß acts in concert with anti-inflammatory cytokines, in particular, IL-1R antagonist (IL-1Ra), to ensure the correct development and outcome of the inflammation: imbalance in the IL-1ß/IL-1Ra ratio is implicated in many human diseases and may lead to dramatic consequences. In this article, we show that single TLR engagement induces IL-1ß and, with a little delay, IL-1Ra. Differently, costimulation of TLR2, TLR4, and TLR7/8 enhances IL-1ß secretion but severely inhibits IL-1Ra production. The IL-1ß/IL-1Ra unbalance after activation of multiple TLRs depends on the insurgence of oxidative stress, because of enhanced production of reactive oxygen species and failure of the antioxidant systems. Increased reactive oxygen species levels increase ATP externalization by monocytes, resulting in enhanced inflammasome activation and IL-1ß secretion. Oxidative stress then induces cell responses to stress, including inhibition of protein synthesis, which, in turn, is responsible for the impaired production of IL-1Ra. IL-1Ra secretion is restored by exogenous antioxidants that oppose oxidative stress. Similar effects are evident also on other cytokines: TNF-α is induced, whereas IL-6 is inhibited by costimulation. Our findings provide a molecular basis to the imbalance between proinflammatory and regulatory cytokine circuits that occur in various pathologic conditions, and suggest new strategies for controlling inflammation.

The secretion of IL-1ß and options for release.

Unlike most cytokines, IL-1ß lacks a secretory signal sequence raising the question of how is this cytokine processed and delivered outside the producing cells. After the seminal observation that IL-1ß is actively secreted by human monocytes through a route alternative to the classic endoplasmic reticulum-Golgi, several different pathways have been proposed for IL-1ß secretion in different cell types and culture conditions, some of which are unique to macrophage cell lines. Here we describe the most credited of these pathways. In particular, we will focus on IL-1ß secretion from primary human blood monocytes. In fact, although data from macrophages or macrophage cell lines are predominant, secretion of IL-1ß by monocytes is the most clinically relevant.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BACKGROUND: CTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma. METHODS: CTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student's t-test. RESULTS: All melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts. CONCLUSIONS: Our studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.