Multiparametric immune profiling to predict the risk of cancer development in chronic immune suppressed solid organ transplant patients

Solid organ transplantation (SOT) is currently recognized as the treatment of choice of patients with end-stage disease, but the length and type of pharmacologic immunosuppression strongly affect patients\u2019 immune system resulting in enhanced immune evasion strategies, oncogenic virus reactivation and malignant transformation. On these grounds, a more in depth characterization of functional defects of immune cells related to the different immunosuppressive regimens and the occurrence of relevant clinical events might be relevant to design novel and integrated cancer surveillance practices for immune suppressed transplant recipients. The main purpose of this study is to identify a reliable immunologic profile predictive of cancer development and progression in SOT patients through a multiparametric phenotypic and functional characterization of dendritic cells (DCs) and T lymphocytes in SOT patients treated with different immunosuppressive regimens. In particular, the activation status of these cells was assessed in serial samples through Multispectral imaging flow cytometry, whereas the extent of T-cell responses specific for pathogenic viruses and tumor-associated antigens was measured by ELIspot IFN-\u3b3 assay. Data obtained in SOT patients who developed a tumor (n=15) were compared with those from patients tumor-free during the follow-up (n=58). Interestingly, we observed that patients experiencing cancer onset during surveillance had lower numbers of activated CD4+ and CD8+ T lymphocytes and a decreased activation of monocyte-derived DCs as compared with the \u201cno tumor\u201d cohort and healthy donors. Moreover, we found that tumor onset patients display higher spontaneous T-cells responses against the \u201cuniversal\u201d tumor associated-antigens hTERT and Survivin if compared to \u201cno tumor\u201d patients and healthy controls. Globally, patients of the \u201ctumor\u201d cohort showed significantly higher levels of circulating hTERT mRNA as compared to those of the \u201cno tumor\u201d cohort, even before the diagnosis of cancer, suggesting that this biomarker could usefully complement the immunomonitoring of SOT patients and help identify those at risk of cancer. We also found that the levels of HPV-specific T-cell responses observed in both patient cohorts were significantly lower both at baseline and at follow up as compared to those of healthy donors, whereas no difference was detected with regard to T-cell responses specific for CMV and EBV. Given the critical role of severe immunosuppression that characterizes transplanted patients and the importance of immune surveillance integrity in the prevention and control of malignancies, the results of the present study provide a strong rationale to design and activate a prospective study in a larger cohort of SOT patients aimed at validating the role of the immune biomarkers identified as potential predictors of the risk of cancer in the highly heterogeneous setting of SOT patients

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages' maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment

Serum Albumin Is Inversely Associated With Portal Vein Thrombosis in Cirrhosis

We analyzed whether serum albumin is independently associated with portal vein thrombosis (PVT) in liver cirrhosis (LC) and if a biologic plausibility exists. This study was divided into three parts. In part 1 (retrospective analysis), 753 consecutive patients with LC with ultrasound-detected PVT were retrospectively analyzed. In part 2, 112 patients with LC and 56 matched controls were entered in the cross-sectional study. In part 3, 5 patients with cirrhosis were entered in the in vivo study and 4 healthy subjects (HSs) were entered in the in vitro study to explore if albumin may affect platelet activation by modulating oxidative stress. In the 753 patients with LC, the prevalence of PVT was 16.7%; logistic analysis showed that only age (odds ratio [OR], 1.024; P = 0.012) and serum albumin (OR, -0.422; P = 0.0001) significantly predicted patients with PVT. Analyzing the 112 patients with LC and controls, soluble clusters of differentiation (CD)40-ligand (P = 0.0238), soluble Nox2-derived peptide (sNox2-dp; P < 0.0001), and urinary excretion of isoprostanes (P = 0.0078) were higher in patients with LC. In LC, albumin was correlated with sCD4OL (Spearman's rank correlation coefficient [r(s)], -0.33; P < 0.001), sNox2-dp (r(s), -0.57; P < 0.0001), and urinary excretion of isoprostanes (r(s), -0.48; P < 0.0001) levels. The in vivo study showed a progressive decrease in platelet aggregation, sNox2-dp, and urinary 8-iso prostaglandin F2 alpha-III formation 2 hours and 3 days after albumin infusion. Finally, platelet aggregation, sNox2-dp, and isoprostane formation significantly decreased in platelets from HSs incubated with scalar concentrations of albumin. Conclusion: Low serum albumin in LC is associated with PVT, suggesting that albumin could be a modulator of the hemostatic system through interference with mechanisms regulating platelet activation

Dissecting the multiplicity of immune effects of immunosuppressive drugs to better predict the risk of de novo malignancies in solid organ transplant patients

De novo malignancies constitute an emerging cause of morbidity after solid organ transplant (SOT), significantly affecting the long-term survival of transplant recipients. Pharmacologic immunosuppression may functionally impair the immunosurveillance in these patients, thereby increasing the risk of cancer development. Nevertheless, the multiplicity and heterogeneity of the immune effects induced by immunosuppressive drugs limit the current possibilities to reliably predict the risk of de novo malignancy in SOT patients. Therefore, there is the pressing need to better characterize the immune dysfunctions induced by the different immunosuppressive regimens administered to prevent allograft rejection to tailor more precisely the therapeutic schedule and decrease the risk of de novo malignancies. We herein highlight the impact exerted by different classes of immunosuppressants on the most relevant immune cells, with a particular focus on the effects on dendritic cells (DCs), the main regulators of the balance between immunosurveillance and tolerance

Altered plasma levels of apixaban in major gastrointestinal tract surgery: A case report and review of the literature

Altered direct oral anticoagulant (DOAC) plasma levels can lead either to spontaneous hemorrhagic or thrombotic complications. We describe a case of suspected altered apixaban disposition in a patient with an upper gastrointestinal cancer resection treated with apixaban for non-valvular atrial fibrillation. Diagnosis of ischemic stroke for left hemiparesis was confirmed due to recent emergence of a hypodense area in the posterior capsular nucleus of ischemic reference in a context of binuclear capsular lacunar lesions. Thus, apixaban underexposure was suspected from anamnestic data and oral anticoagulation was switched to parenteral at the next scheduled dose for stroke recurrence. Before switching apixaban pharmacokinetic analysis was performed and unexpectedly showed apixaban plasma overexposure. After 3 days from the switch, the patient experienced spontaneous bleeding complications, for which the risk-benefit profile of continuing anticoagulant treatment for stroke recurrences warranted treatment discontinuation. Unexpected DOAC plasma exposure may present in special patient populations with thrombotic and bleeding complications. Though universally recognized therapeutic ranges have yet to be established for DOACs, periodic drug monitoring may aid in guiding optimization of DOAC therapy and reduce the risk of adverse events in special patient populations

IL-10 and ARG-1 Concentrations in Bone Marrow and Peripheral Blood of Metastatic Neuroblastoma Patients Do Not Associate with Clinical Outcome

The expression of the immunosuppressive molecules IL-10 and arginase 1 (ARG-1), and of FOXP3 and CD163, as markers of regulatory T cells (Treg) and macrophages, respectively, was evaluated in bone marrow (BM) and peripheral blood (PB) samples collected at diagnosis from patients with metastatic neuroblastoma (NB). IL-10 and ARG-1 plasma concentrations were measured and the association of each parameter with patients’ outcome was tested. The percentages of immunosuppressive Treg and type-1 regulatory (Tr1) cells were also determined. In both BM and PB samples, IL-10 mRNA expression was higher in metastatic NB patients than in controls. IL-10 plasma concentration was higher in patients with NB regardless of stage. Neither IL-10 expression nor IL-10 plasma concentration significantly associated with patient survival. In PB samples from metastatic NB patients, ARG-1 and CD163 expression was higher than in controls but their expression did not associate with survival. Moreover, ARG-1 plasma concentration was lower than in controls, and no association with patient outcome was found. Finally, in metastatic NB patients, the percentage of circulating Treg was higher than in controls, whereas that of Tr1 cells was lower. In conclusion, although IL-10 concentration and Treg percentage were increased, their contribution to the natural history of metastatic NB appears uncertain

Radical Hemithoracic Radiotherapy Induces Systemic Metabolomics Changes That Are Associated with the Clinical Outcome of Malignant Pleural Mesothelioma Patients

Simple SummaryRadical hemithoracic radiotherapy represents a promising new advance in the field of radiation oncology and encouraging results have been achieved in the treatment of malignant pleural mesothelioma patients. This study showed that this radiotherapy modality produces significant changes in serum metabolomics profile mainly affecting arginine and polyamine biosynthesis pathways. Interestingly, individual metabolomics alterations were found associated with the clinical overall survival outcome of the radiotherapy treatment. These results highlight metabolomics profile analysis as a powerful prognostic tool useful to better understand the mechanisms underlying the interpatients variability and to identify patients who may receive the best benefit from this specific radiotherapy treatment.Radical hemithoracic radiotherapy (RHRT) represents an advanced therapeutic option able to improve overall survival of malignant pleural mesothelioma patients. This study aims to investigate the systemic effects of this radiotherapy modality on the serum metabolome and their potential implications in determining the individual clinical outcome. Nineteen patients undergoing RHRT at the dose of 50 Gy in 25 fractions were enrolled. Serum targeted metabolomics profiles were investigated at baseline and the end of radiotherapy by liquid chromatography and tandem mass spectrometry. Univariate and multivariate OPLS-DA analyses were applied to study the serum metabolomics changes induced by RHRT while PLS regression analysis to evaluate the association between such changes and overall survival. RHRT was found to affect almost all investigated metabolites classes, in particular, the amino acids citrulline and taurine, the C14, C18:1 and C18:2 acyl-carnitines as well as the unsaturated long chain phosphatidylcholines PC ae 42:5, PC ae 44:5 and PC ae 44:6 were significantly decreased. The enrichment analysis showed arginine metabolism and the polyamine biosynthesis as the most perturbed pathways. Moreover, specific metabolic changes encompassing the amino acids and acyl-carnitines resulted in association with the clinical outcome accounting for about 60% of the interpatients overall survival variability. This study highlighted that RHRT can induce profound systemic metabolic effects some of which may have a significant prognostic value. The integration of metabolomics in the clinical assessment of the malignant pleural mesothelioma could be useful to better identify the patients who can achieve the best benefit from the RHRT treatment

Identification of rare alleles in an Italian population of 284 patients with 21- hydroxylase deficiency by complete sequencing of the CYP21 gene

The screening of the usually tested CYP21 gene alterations (the large gene deletion/conversion and the P30L, IVS2-13A/C>G, Ex 3 D8nt, Ex6 cluster, I172N, V281L, 1766-1767insT, Q318X, R356Q, P453S) by means of Southern blotting and Allele-Specific PCR in an Italian population of 284 patients with 21-hydroxylase deficiency led us to characterize 461 of the total 506 different alleles (91.1%), while 45 (8,9 %) remained uncharacterized. Some incongruent genotype/phenotype correlation was also observed. In order to reduce the number of uncharacterized alleles and possibly to identify additional alterations in some patients’ alleles, we reanalysed the entire population (128 classic and 156 nonclassic forms selected for stimulated 17-HP level>10 ng/ml) by complete sequencing of the CYP21 gene, promoter included. Identified mutations were verified in the available parents to confirm allele segregation. Result a: of the 461 characterized alleles, 10 present an additional/different known mutation from that previously identified (2.1% false positive/negative results); 7 showed a conversion extending from the promoter to the P30L mutation (3 cases) or the IVS2 (4 cases). Result b: of the 34 uncharacterized alleles, 16 showed rare mutations (1 M283V, 1 R316X, 4 R341P, 4 R356Q, 1 R426H, 4 P482S, 1 R483P); 7 showed as far as we know unique mutations, 2 (W19X, L480Xfs) with an obvious implication in the phenotype, and 5 affecting residues L142, I171, R341, V358, L446 currently being studied; 11 remained uncharacterized. A total of 43 (23 of pseudogene origin) SNPs, 15 in the promoter, 3 in the 3’ UTR and 25 in introns were identified: further studies will verify their frequency in controls and the possible implication in the phenotypes. The high frequence of complex, rare or unique alleles in the Italian population underlines the importance to routinely analyse the CY21 gene by complete sequencing to avoid false/incomplete results, identify new mutations or sequence variations and to improve the genotype/phenotype correlation, genetic counselling and treatment