Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients.

BACKGROUND: Tregs trafficking is controlled by CXCR4. In Renal Cell Carcinoma (RCC), the effect of the new CXCR4 antagonist, R54, was explored in peripheral blood (PB)-Tregs isolated from primary RCC patients. METHODS: PB-Tregs were isolated from 77 RCC patients and 38 healthy donors (HDs). CFSE-T effector-Tregs suppression assay, IL-35, IFN-γ, IL-10, TGF-ß1 secretion, and Nrp-1+Tregs frequency were evaluated. Tregs were characterised for CTLA-4, PD-1, CD40L, PTEN, CD25, TGF-ß1, FOXP3, DNMT1 transcriptional profile. PTEN-pAKT signalling was evaluated in the presence of R54 and/or triciribine (TCB), an AKT inhibitor. Methylation of TSDR (Treg-Specific-Demethylated-Region) was conducted. RESULTS: R54 impaired PB-RCC-Tregs function, reduced Nrp-1+Tregs frequency, the release of IL-35, IL-10, and TGF-ß1, while increased IFN-γ Teff-secretion. The CXCR4 ligand, CXCL12, recruited CD25+PTEN+Tregs in RCC while R54 significantly reduced it. IL-2/PMA activates Tregs reducing pAKT+Tregs while R54 increases it. The AKT inhibitor, TCB, prevented the increase in pAKT+Tregs R54-mediated. Moreover, R54 significantly reduced FOXP3-TSDR demethylation with DNMT1 and FOXP3 downregulation. CONCLUSION: R54 impairs Tregs function in primary RCC patients targeting PTEN/PI3K/AKT pathway, reducing TSDR demethylation and FOXP3 and DNMT1 expression. Thus, CXCR4 targeting is a strategy to inhibit Tregs activity in the RCC tumour microenvironment.

Potential clinical implications of CD4+CD26high T cells for nivolumab treated melanoma patients.

BACKGROUND: Nivolumab is an anti-PD1 antibody that has dramatically improved metastatic melanoma patients' outcomes. Nevertheless, many patients are resistant to PD-1 inhibition, occasionally experiencing severe off-target immune toxicity. In addition, no robust and reproducible biomarkers have yet been validated to identify the correct selection of patients who will benefit from anti-PD-1 treatment avoiding unwanted side effects. However, the strength of CD26 expression on CD4+ T lymphocytes permits the characterization of three subtypes with variable degrees of responsiveness to tumors, suggesting that the presence of CD26-expressing T cells in patients might be a marker of responsiveness to PD-1-based therapies. METHODS: The frequency distribution of peripheral blood CD26-expressing cells was investigated employing multi-parametric flow cytometry in 69 metastatic melanoma patients along with clinical characteristics and blood count parameters at baseline (W0) and compared to 20 age- and sex-matched healthy controls. Percentages of baseline CD4+CD26high T cells were correlated with the outcome after nivolumab treatment. In addition, the frequency of CD4+CD26high T cells at W0 was compared with those obtained after 12 weeks (W1) of therapy in a sub-cohort of 33 patients. RESULTS: Circulating CD4+CD26high T cells were significantly reduced in melanoma patients compared to healthy subjects (p = 0.001). In addition, a significant association was observed between a low baseline percentage of CD4+CD26high T cells (< 7.3%) and clinical outcomes, measured as overall survival (p = 0.010) and progression-free survival (p = 0.014). Moreover, patients with clinical benefit from nivolumab therapy had significantly higher frequencies of circulating CD4+CD26high T cells than patients with non-clinical benefit (p = 0.004) at 12 months. Also, a higher pre-treatment proportion of circulating CD4+CD26high T cells was correlated with Disease Control Rate (p = 0.014) and best Overall Response Rate (p = 0.009) at 12 months. Interestingly, after 12 weeks (W1) of nivolumab treatment, percentages of CD4+CD26high T cells were significantly higher in comparison with the frequencies measured at W0 (p < 0.0001), aligning the cell counts with the ranges seen in the blood of healthy subjects. CONCLUSIONS: Our study firstly demonstrates that peripheral blood circulating CD4+CD26high T lymphocytes represent potential biomarkers whose perturbations are associated with reduced survival and worse clinical outcomes in melanoma patients.

Exosomal CD73 from serum of patients with melanoma suppresses lymphocyte functions and is associated with therapy resistance to anti-PD-1 agents.

BACKGROUND: CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here, we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy. METHODS: Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry, also in the presence of selective inhibitors of CD73. Interferon (IFN)-γ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control. RESULTS: Isolated exosomes carried CD73 on their surface, which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-γ release, which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment, expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders, while exosomal CD73 resulted significantly increased in non-responders. CONCLUSIONS: CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

Serum CD73 is a prognostic factor in patients with metastatic melanoma and is associated with response to anti-PD-1 therapy.

BACKGROUND: Inhibitors of immune checkpoint programmed cell death protein 1 (PD-1) receptor on T cells have shown remarkable clinical outcomes in metastatic melanoma. However, most patients are resistant to therapy. Production of extracellular adenosine, via CD73-mediated catabolism of AMP, contributes to suppress T-cell-mediated responses against cancer. In this study, we analyzed the expression and activity of soluble CD73 in sera of patients with melanoma undergoing anti-PD-1± cytotoxic T-lymphocyte-associated antigen 4 therapy. METHODS: Soluble CD73 expression and activity were retrospectively analyzed in serum of a total of 546 patients with melanoma from different centers before starting treatment (baseline) with anti-PD-1 agents, nivolumab or pembrolizumab, and compared with those of 96 healthy subjects. The CD73 activity was correlated with therapy response and survival of patients. RESULTS: Patients with melanoma show significantly higher CD73 activity and expression than those observed in healthy donors (p<0.0001). Elevated pretreatment levels of CD73 activity were associated with non-response to therapy with nivolumab or pembrolizumab. During treatment, levels of soluble CD73 activity remain unchanged from baseline and still stratify clinical responders from non-responders. High levels of serum CD73 enzymatic activity associate with reduced overall survival (OS; HR=1.36, 95% CI 1.03 to 1.78; p=0.03) as well as progression-free survival (PFS; HR=1.42, 95% CI 1.13 to 1.79, p=0.003). Further, the multivariate Cox regression analysis indicates that serum CD73 activity is an independent prognostic factor besides serum lactate dehydrogenase levels and the presence of brain metastases for both OS (p=0.009) and PFS (p=0.001). CONCLUSION: Our data indicate the relevance of serum CD73 in patients with advanced melanoma receiving anti-PD-1 therapy and support further investigation on targeting CD73 in combination with anti-PD-1 antibodies.

Inhibiting Monocyte Recruitment to Prevent the Pro-Tumoral Activity of Tumor-Associated Macrophages in Chondrosarcoma.

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH2, denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice.

Targeted sequencing analysis of cell-free DNA from metastatic non-small-cell lung cancer patients: clinical and biological implications.

BACKGROUND: Sequencing artifacts, clonal hematopoietic mutations of indeterminate potential (CHIP) and tumor heterogeneity have been hypothesized to contribute to the low concordance between tissue and cell-free DNA (cfDNA) molecular profiling with targeted sequencing. METHODS: We analyzed by targeted sequencing cfDNA from 30 healthy individuals, and cfDNA and matched tumor samples from 30 EGFR-mutant and 77 EGFR wild-type metastatic non-small-cell lung cancer (mNSCLC) patients. Discordant cases were solved by droplet digital PCR (ddPCR). RESULTS: By testing cfDNA from healthy donors, we developed an algorithm to recognize sequencing artifacts. Applying this method to cfDNA from mNSCLC patients, EGFR mutations were detected with a good sensitivity (76.7%) and specificity (97.4%). In contrast, sensitivity and specificity for KRAS variants were 61.5% and 93.8%, respectively. All EGFR and KRAS variants detected in plasma but not in tissue were confirmed by ddPCR, thus excluding sequencing artifacts. In a fraction of cases, KRAS mutations found in plasma samples were confirmed in tumor tissue suggesting tumor heterogeneity. KRAS variants were found to be more likely sub-clonal as compared with EGFR mutations, and a correlation between clonal origin and frequency of detection in plasma was found. In a case with both EGFR and KRAS variants in cfDNA, we could demonstrate the presence of the KRAS variant in tumor tissue associated with lack of response to tyrosine kinase inhibitors (TKIs). CONCLUSIONS: Although sequencing artifacts can be identified in targeted sequencing of cfDNA, tumor heterogeneity and CHIP are likely to influence the concordance between plasma and tissue testing.

Frequency of circulating CD8+CD73+T cells is associated with survival in nivolumab-treated melanoma patients.

BACKGROUND: PD-1 blocking agents, such as nivolumab, have demonstrated clear anti-tumor effects and clinical benefits in a subset of patients with advanced malignancies. Nonetheless, more efforts are needed to identify reliable biomarkers for outcome, to correctly select patients who will benefit from anti-PD-1 treatment. The aim of this study was to investigate the role of peripheral CD8+T cells expressing CD73, involved in the generation of the immune suppressive molecule adenosine, in predicting outcome after nivolumab treatment in advanced melanoma patients. METHODS: PBMCs from 100 melanoma patients treated with nivolumab were collected at National Cancer Institute "G. Pascale" of Naples. Frequencies of CD8+ lymphocytes phenotypes were assessed by flow cytometry at baseline before nivolumab treatment, along with clinical characteristics and blood count parameters. Healthy controls (n = 20) were also analysed. Percentages of baseline T cells expressing PD-1 and CD73 were correlated with outcome after nivolumab treatment. RESULTS: Melanoma patients presented a lower frequency of total circulating CD8+ lymphocytes than control subjects (p = 0.008). Patients with low baseline percentage of circulating CD8+PD-1+CD73+ lymphocytes (< 2.3%) had better survival (22.4 months vs 6.9 months, p = 0.001). Patients (39%) with clinical benefit from nivolumab therapy presented a significantly lower frequency of circulating CD8+PD-1+CD73+ lymphocytes than patients who progressed to nivolumab treatment (p = 0.02). CONCLUSIONS: Our observations suggest that baseline CD73 expression on circulating CD8+PD-1+ lymphocytes appear a promising biomarker of response to anti-PD-1 treatment in melanoma patients. Further investigations are needed for validation and for clarifying its role as prognostic or predictive marker.

Correction: Nine-year distribution pattern of hepatitis C virus (HCV) genotypes in Southern Italy.

[This corrects the article DOI: 10.1371/journal.pone.0212033.].

Nine-year distribution pattern of hepatitis C virus (HCV) genotypes in Southern Italy.

INTRODUCTION: It has been greatly described that different hepatitis C virus (HCV) genotypes are strictly correlated to various evolution, prognosis and response to therapy during the chronic liver disease. Aim of this study was to outline the changes in the epidemiology of Hepatitis C genotypes in Southern Italy regions from 2006 to 2014. MATERIAL/METHODS: Prevalence of HCV genotypes was analyzed in 535 HCV-RNA positive patients with chronic Hepatitis C infection, selected during the period 2012-2014, and compared with our previous data, referred to periods 2006-2008 and 2009-2011. RESULTS: In all the three periods analyzed, genotype 1b is predominant (51.8% in 2006-08, 48.3% in 2009-11 and 54.4% in 2012-14) while genotype 2 showed an increase in prevalence (27.9% in 2006-08, 31.7% in 2009-11 and 35.2% in 2012-14) and genotypes 3a and 1a a decrease during the same period (6.8% in 2006-08, 4.7% in 2009-11 and 3.2% in 2012-14 and 7.9% in 2006-08, 4.7% in 2009-11 and 2.6% in 2012-14, respectively). Subtype 1b seems to be equally distributed between males and females (52.7% vs 56.6%) and the prevalence in the age range 31-40 years is significantly higher in the 2012-14 period than in both previous periods (53.8% vs. 16.6% in 2009-11, p< 0.001 and 13.4% in 2006-08, p < 0.001). CONCLUSIONS: Genotype 1b is still the most prevalent, even if shows a significantly increase in the under 40 years old population. Instead, genotype 3a seems to have a moderate increase among young people. Overall, the alarming finding is the "returning" role of the iatrogenic transmission as risk factor for the diffusion of Hepatitis C infection.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges.

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.

Autologous Platelet Gel Improves Bone Reconstruction of Large Defects in Patients with Bone Giant Cell Tumors.

BACKGROUND/AIM: Giant cell tumors are mostly benign but locally aggressive tumors. The excision of bone tumors can result in large defects, therefore bone reconstruction is still one the most demanding procedures in orthopedic surgery. Our study addresses the opportunity for improving surgical outcome by employing ß-tricalcium phosphate (ß-TCP) with platelet-rich plasma (PRP) at the surgical site. PATIENTS AND METHODS: We included 16 patients with giant cell tumors. After adjuvant therapy, the cavity was reconstructed with ß-TCP, bone graft material (ActifuserR Granules Baxter) and platelet gel application. RESULTS: Our explorative analysis suggests a positive effect of PRP on surgical outcome in patients with giant cell tumors treated with curettage. CONCLUSION: Use of platelet gel as an adjuvant significantly reduces the time required for bone healing following intralesional treatment of benign giant cell tumors, and achieves good functional results without promoting local recurrence.

Distribution pattern of hepatitis C virus genotypes and correlation with viral load and risk factors in chronic positive patients.

OBJECTIVE: Hepatitis C virus (HCV) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. The purpose of this study was to describe the distribution pattern of HCV genotypes in chronic hepatitis patients in the Campania region of southern Italy and estimate their association with risk factors and viral load. MATERIALS AND METHODS: 404 consecutive HCV ribonucleic acid-positive patients were included in the study. HCV genotyping was carried out by the HCV line probe assay test and viral load estimation by the TaqMan real-time PCR system. RESULTS: The predominant genotype was 1 (63.6%), followed by genotype 2 (29.4%), 3 (6.2%) and 4 (0.8%). Subtype 1b was more frequent in females than in males. Conversely, genotype 3 was more frequent in males. No significant difference was observed in age distribution of HCV genotypes. Surgery and dental therapy were the most frequent risk factors for genotype 1 and intravenous drug abuse and tattooing for genotype 3. Patients with genotype 1 more frequently showed high HCV viral load when compared to those with genotypes 2 and 3. CONCLUSION: The present study revealed that HCV genotypes 1 and 2 accounted for over 95% of all HCV infections in the Campania region, and genotype 1 was more frequently associated with a higher viral load when compared to genotypes 2 and 3.

HBV transmission from an occult carrier with five mutations in the major hydrophilic region of HBsAg to an immunosuppressed plasma recipient.

We describe the case of transmission of an HBsAg-negative hepatitis B infection to an immunosuppressed patient by plasma donation from an HBsAg-negative subject, but with very low serum HBV DNA (about 50 IU/ml) and five mutations in the major hydrophilic region of HBsAg.

Age and gender distribution of hepatitis C virus genotypes in the metropolitan area of Naples.

OBJECTIVE: To assess any time-related variations in the distribution of hepatitis C virus (HCV) genotypes in the metropolitan area of Naples, Italy. METHODS: HCV genotypes were analysed in 255 HCV RNA-positive patients with chronic HCV infection, observed from 2009 to 2011, and compared with data pertaining to a sample of 176 HCV RNA-positive patients observed from 2006 to 2008. RESULTS: In both periods of analysis, genotype 1b was predominant (51.8 and 48.3%, respectively), followed by genotype 2 (27.9 and 31.7%, respectively). These HCV genotypes were particularly prevalent in older patients, whereas genotypes 3a and 1a were observed more frequently in the younger population. Genotype 1b was particularly common in females compared to males in both study periods (39.3% of 89 males vs. 64.3% of 87 females in the 3-year period 2006-2008, p < 0.001; 41.5% of 118 males vs. 54.0% of 137 females in the period 2009-2011, p < 0.05). The prevalence of patients with genotype 1b in the age range 51-60 years was higher in the 2006-2008 period than in 2009-2011 (76.9 vs. 37.7%; p < 0.0005) and lower in the over 60 year olds (55.1 vs. 59.6%; p = 0.5). CONCLUSION: Genotype 1b, historically the most prevalent in Italy, is still predominant; however, when comparing the two time periods, a cohort effect evidencing the increasing prevalence of genotype 1b among elderly patients was revealed.

Platelet gel in cutaneous radiation dermatitis.

INTRODUCTION: Radiotherapy, alone or in combination with chemotherapy and/or surgery, is a fundamental and irreplaceable method of treating tumours. Nonetheless, although the technological advances made during recent years and the associated improvements in this type of treatment have reduced the incidence of complications, 5-15 % of patients still experience damage to the healthy tissues exposed to radiation. Cutaneous and mucosal lesions are severe collateral effects of radiotherapy that have an enormous impact on a patient's quality of life. Unfortunately, however, the efficacy of conventional treatments, while demonstrably useful in acute lesions, remains disputed in chronic cases. Nevertheless, numerous studies and clinical findings have demonstrated that topical, non-transfusional plasma-rich platelet gel is able to accelerate the regeneration and repair of tissues through the action of the various growth factors contained within the alpha granules of platelets. We therefore set out to evaluate the efficacy of autologous platelet gel, chosen for its limited cost and ease of preparation, in chronic cutaneous radiation dermatitis. METHODS: "Home-made" platelet gel was produced by treating platelets with autologous thrombin. The safety of the product was ensured by microbiological tests. The autologous platelet gel was applied topically once a week, for a mean duration of 35 days, to chronic third- and fourth-degree (European Pressure Ulcer Advisory Panel classification and Common Terminology Criteria for Adverse Events score) cutaneous radiation dermatitis in a group of ten patients previously treated for moderate-to-high grade (histology G2-G3) limb sarcoma by tumour excision and post-surgical radiotherapy (dose 50-64 Gy). The radiation dermatitis had appeared at different intervals after treatment and had all proved resistant to conventional treatments. RESULTS: The autologous platelet gel was found to be successful in seven out of the ten patients treated. The various phases of the healing process were observed in all cases. Platelet gel application was suspended in three patients: in one patient after one application due to tumour progression, in another patient after two applications due to development of distant metastases and in the third after six applications with only partial tissue response. At 5-year follow-up, six of the seven successfully treated patients remained free of both disease and lesion, while the remaining patient, the eldest, had passed away in the interim due to extraneous causes. CONCLUSION: Platelet gel treatment could therefore be used to bring about healing in chronic cutaneous radiation dermatitis, lending itself to better patient compliance and a favourable cost/benefit ratio, due to a reduction in the number of medications and hospital visits required.