Early serum TARC reduction predicts prognosis in advanced-stage Hodgkin lymphoma patients treated with a PET-adapted strategy.

Among patients with advanced-stage classical Hodgkin lymphoma (cHL) receiving ABVD chemotherapy, PET performed after the first two treatment cycles (PET-2) has prognostic value. However, 15% of patients with a negative PET-2 will experience treatment failure. Here we prospectively evaluated serum thymus and activation-regulated chemokine (TARC) levels, to improve risk assessment in patients treated according to HD0607 PET-driven trial (#NCT00795613). In 266 patients with available serum samples, who have agreed to participate in a sub-study for assessment of the role of TARC monitoring, serum TARC levels were measured at baseline and at time of PET-2 by commercially available ELISA test kits. The primary end-point was to evaluate the association between TARC after 2 ABVD cycles and PFS. Median TARC-2 values were significantly higher in PET-2-positive patients compared to PET-2-negative patients (P = .001), and in patients with treatment failure compared to those in continuous CR (P = .01). The 4-year PFS significantly differed between patients with TARC-2 >800 pg/mL vs ≤800 pg/mL (64% vs 86%, P = .0001). Moreover, among PET-2-negative patients, elevated TARC-2 identified those with a worse prognosis (74% vs 89%; P = .01). In multivariable analysis, TARC-2 >800 pg/mL was a significant independent predictor of PFS in the whole study population (HR 2.39, P = .004) and among the PET-2-negative patients (HR 2.49, P = .02). In conclusion, our results indicate that TARC-2 serum levels above 800 pg/mL suggest the need for a stringent follow-up in PET-2-negative patients, and the evaluation of new drugs in PET-2-positive, who will likely fail to respond to intensification with escalated BEACOPP.

Prolonged activity and toxicity of sirolimus in a patient with metastatic renal perivascular epithelioid cell tumor: a case report and literature review.

Perivascular epithelioid cell tumor (PEComa) is a family of mesenchymal tumors. Conventional chemotherapy has little activity in this disease, but case reports are available on the activity of mammalian target of rapamycin inhibitors (e.g. sirolimus and temsirolimus). Pharmacokinetic assays of sirolimus are available as this drug has a precise therapeutic window and blood levels might be influenced by CYP3A4 polymorphisms and drug interactions. We report on a case of a patient with metastatic, progressive PEComa who started sirolimus at a dose of 5 mg/day with evidence of grade (G) 3 mucositis, G2 thrombocytopenia, and G1 leucopenia 10 days after the treatment started, in absence of concomitant medications or prohibited food assumption. Elevated sirolimus blood levels were detected (156.8 ng/ml). Sirolimus was stopped, and toxicity resolved in 5 weeks. Computed tomography scan 2 months after the treatment started showed a partial response (RECIST). After toxicity resolution, the patient restarted sirolimus at a dose of 1 mg/day, with blood levels in the range of 10-20 ng/ml. Tumor response was confirmed and maintained, and the patient is still under treatment 18 months later, with no additional adverse effects. Genetic analysis of five selected polymorphisms (rs2740574, rs776746, rs1128503, rs2032582, and rs1045642) in drug metabolism enzymes and transporters did not provide a clear explanation of the observed unusual pharmacokinetic. This case confirms the activity of mammalian target of rapamycin inhibitors in PEComa and strengthens the importance of pharmacokinetic drug blood levels monitoring in patients treated with sirolimus. In our patient, after dose adjustment, sirolimus could be restarted with a prolonged clinical benefit and no additional toxicity.

Application of Controlled Shear Stresses on the Erythrocyte Membrane as a New Approach to Promote Molecule Encapsulation.

Human red blood cells (RBCs) have a remarkable capacity to undergo reversible membrane swelling. Resealed erythrocytes have been proposed as carriers and bioreactors to be used in the treatment of various diseases. This work is aimed at developing a setup allowing the encapsulation of test molecules into erythrocytes by inducing reversible pore formation on the RBC membrane through the application of controlled mechanical shear stresses. The designed setup consists of two reservoirs connected by a glass capillary. Each reservoir is connected to a compressor; during the tests, the reservoirs were in turn pressurized to promote erythrocyte flow through the capillary. The setup was filled with a suspension of erythrocytes, phosphate buffer, and FITC-dextran. Dextran was chosen as the diffusive molecule to check membrane pore dimensions. Samples of the suspension were withdrawn at scheduled times while the setup was operating. Flow cytometry and stereo-optical microscopy analyses were used to evaluate the erythrocyte dextran uptake. The setup was shown to be safe, well controlled, and adjustable. The outcomes of the experimental tests showed significant dextran uptake by RBCs up to 8%. Microscopy observations highlighted the formation of echinocytes in the analyzed samples. Erythrocytes from different donors showed different reactions to mechanical stresses. The experimental outcomes proved the possibility to encapsulate test molecules into erythrocytes by applying controlled mechanical shear stresses on the RBC membrane, encouraging further studies.

Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients.

Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T-cell lymphoma, and it is currently used off-label for graft-versus-host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off-line technique. Since no gold-standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty-one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8-methoxy-psoralen plus ultraviolet A; the variation (ΔAPOPTOSIS ) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, ΔAPOPTOSIS was >15% (the "alerting" threshold for ΔAPOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test-being faster and easier than the proliferation test-could be a reliable way to validate ECP procedures.

Serum thymus and activation-regulated chemokine level monitoring may predict disease relapse detected by PET scan after reduced-intensity allogeneic stem cell transplantation in patients with Hodgkin lymphoma.

Patients with relapsed and refractory Hodgkin lymphoma (HL) may experience long-term survival after allogeneic stem cell transplantation (alloSCT), but disease recurrence represents the main cause of treatment failure. Positron-emission tomography (PET)-positive patients after alloSCT have a dismal outcome. Serum thymus and activation-regulated chemokine (TARC) is produced by Reed-Sternberg cells and may be a marker of disease. Our study aimed at assessing whether TARC levels after alloSCT correlated with disease status and whether TARC monitoring could increase the ability to predict relapse. Twenty-four patients were evaluated in a prospective observational study. TARC serum level and PET were assessed before and after alloSCT during the follow-up (median, 30 months; range, 2 to 54). Before alloSCT, the median TARC level was 721 pg/mL (range, 209 to 1332) in PET-negative patients and 2542 pg/mL (range, 94 to 13,870) in PET-positive patients. After alloSCT, TARC was 620 pg/mL (range, 12 to 4333) in persistently PET-negative patients compared with 22,397 pg/mL (range, 602 to 106,578) in PET-positive patients (P < .0001). In 7 patients who relapsed after alloSCT, TARC level increased progressively even before PET became positive, with a median fold increase of 3.19 (range, 1.66 to 7.11) at relapse. The cut-off value of 1726 pg/mL had a sensitivity of 100% and a specificity of 71% for PET positivity. Patients with at least 1 TARC value above 1726 pg/mL during the first year after alloSCT had a worse progression-free survival (P = .031). In conclusion, TARC was correlated with disease status and its monitoring may be able to predict PET positivity after alloSCT, thus potentially allowing an early immune manipulation.

Identification of the novel HLA-C allele, HLA-C*04:520.

HLA-C*04:520, a novel HLA-C allele, differs from HLA-C*04:01:01 by one mismatch in exon 5.

Comparing BamHI-W and CE-marked assays to detect circulating Epstein-Barr Virus (EBV) DNA of nasopharyngeal cancer patients in a non-endemic area.

OBJECTIVES: Plasma Epstein-Barr Virus (EBV)-DNA is a well-established prognostic biomarker in nasopharyngeal carcinoma (NPC). Different methods for assessment include single-copy gene targeted, European Conformity (CE)-marked assays, which are mostly employed in non-endemic settings, vs multiple-copy gene targeted, in-house BamHI-W based assays, which currently represent the most widely used method for EBV-DNA quantification. To date, evidence concerning the commutability of these different assays is still limited. MATERIALS AND METHODS: From August 2016 to March 2018, 124 plasma and 124 whole blood (WB) samples from 93 NPC patients were collected at different time-points for each patient. EBV-DNA viral load was quantified in pre- (n = 12) and post-treatment (n = 9), follow-up (n = 53), and recurrent/metastatic (R/M) (n = 50) phase. For each sample, one in-house BamHI-W vs three different CE-marked plasma assays were compared; the performance of plasma vs WB matrix was also assessed. Quantitative agreement of EBV-DNA values was evaluated by linear correlation and Bland-Altman analysis. RESULTS: A statistically significant (p = 0.0001) agreement between all CE-marked and the BamHI-W assays was found using plasma matrix, regardless of clinical phase. The results obtained in copies/ml were comparable to those expressed in IU/ml. When using WB matrix, the number of positive detections increased in the post-treatment phase. CONCLUSIONS: Our retrospective comparison supported an agreement between Plasma BamHI-W and CE-marked assays in measuring EBV-DNA for non-endemic NPC patients. There were no significant interferences from different measurement units (IU/ml vs copies/ml). Further evaluations are needed to better clarify the role of WB.

Shear-Induced Encapsulation into Red Blood Cells: A New Microfluidic Approach to Drug Delivery.

Encapsulating molecules into red blood cells (RBCs) is a challenging topic for drug delivery in clinical practice, allowing to prolong the residence time in the body and to avoid toxic residuals. Fluidic shear stress is able to temporary open the membrane pores of RBCs, thus allowing for the diffusion of a drug in solution with the cells. In this paper, both a computational and an experimental approach were used to investigate the mechanism of shear-induced encapsulation in a microchannel. By means of a computational fluid dynamic model of a cell suspension, it was possible to calculate an encapsulation index that accounts for the effective shear acting on the cells, their distribution in the cross section of the microchannel and their velocity. The computational model was then validated with micro-PIV measurements on a RBCs suspension. Finally, experimental tests with a microfluidic channel showed that, by choosing the proper concentration and fluid flow rate, it is possible to successfully encapsulate a test molecule (FITC-Dextran, 40 kDa) into human RBCs. Cytofluorimetric analysis and confocal microscopy were used to assess the RBCs physiological shape preservation and confirm the presence of fluorescent molecules inside the cells.

Induction of both CD8+ and CD4+ T-cell-mediated responses in colorectal cancer patients by colon antigen-1.

PURPOSE: Colon antigen-1 (COA-1) was recently identified as a novel antigen of colorectal cancer encoded by the UBXD5 gene. Here, we evaluated whether a specific T-cell-mediated response directed against this molecule can occur in colorectal cancer patients. EXPERIMENTAL DESIGN: Antigen- and tumor-specific immunologic responses of peripheral blood mononuclear cells (PBMC) stimulated in vitro with the MHC class II-associated immunogenic epitope of COA-1 (FSTFPPTLYQDDTLTLQAAG) were analyzed by IFN-gamma ELISPOT assay. RESULTS: COA-1-specific and tumor-reactive T lymphocytes were isolated from all (n = 7) HLA-DRbeta1*0402+ or *1301+ colorectal cancer patients with progressive disease (Dukes' C and D) but not in patients (n = 4) with early-stage tumor (Dukes' A and B) and in healthy donors (n = 5), suggesting that the immune response against this antigen is associated with the progression of colorectal cancer. COA-1- and tumor-specific T lymphocytes displayed a CD3+CD4+CD69+CD45RA+ phenotype, compatible with the activated effector-type T-cell subset, and most of them exerted cytotoxic activity against HLA-matched and COA-1+ tumor cells. COA-1-specific T cells could also be isolated by in vitro stimulation of peripheral blood mononuclear cells with autologous dendritic cells loaded with tumor lysate, suggesting that this antigen can generate a dominant immunologic response against colorectal cancer cells. Notably, we could identify also COA-1-derived epitopes binding to HLA-A*0201 molecules that elicited antigen- and tumor-specific CD8+ T-cell-mediated responses in colorectal cancer patients. CONCLUSIONS: Both CD4+ and CD8+ T-cell responses against COA-1 can occur in colorectal cancer patients with metastatic disease, suggesting that this antigen is suitable for immunotherapeutic protocols of these patients.

Monitoring Vitamin B12 in Women Treated with Metformin for Primary Prevention of Breast Cancer and Age-Related Chronic Diseases.

Metformin (MET) is currently being used in several trials for cancer prevention or treatment in non-diabetics. However, long-term MET use in diabetics is associated with lower serum levels of total vitamin B12. In a pilot randomized controlled trial of the Mediterranean diet (MedDiet) and MET, whose participants were characterized by different components of metabolic syndrome, we tested the effect of MET on serum levels of B12, holo transcobalamin II (holo-TC-II), and methylmalonic acid (MMA). The study was conducted on 165 women receiving MET or placebo for three years. Results of the study indicate a significant overall reduction in both serum total B12 and holo-TC-II levels according with MET-treatment. In particular, in the MET group 26 of 81 patients and 10 of the 84 placebo-treated subjects had B12 below the normal threshold (<221 pmol/L) at the end of the study. Considering jointly all B12, Holo-TC-II, and MMA, 13 of the 165 subjects (10 MET and 3 placebo-treated) had at least two deficits in the biochemical parameters at the end of the study, without reporting clinical signs. Although our results do not affect whether women remain in the trial, B12 monitoring for MET-treated individuals should be implemented.

Circulating pre-treatment Epstein-Barr virus DNA as prognostic factor in locally-advanced nasopharyngeal cancer in a non-endemic area.

The prognostic value of pre-treatment Epstein-Barr Virus (EBV) DNA viral load for non-endemic, locally-advanced, EBV-related nasopharyngeal cancer (NPC) patients is yet to be defined. All patients with EBV encoded RNA (EBER)-positive NPC treated at our Institution from 2005 to 2014 with chemotherapy (CT) concurrent with radiation (RT) +/- induction chemotherapy (ICT) were retrospectively reviewed. Pre-treatment baseline plasma EBV DNA (b-EBV DNA) viral load was detected and quantified by PCR. Median b-EBV DNA value was correlated to potential influencing factors by univariate analysis. Significant variables were then extrapolated and included in a multivariate linear regression model. The same variables, including b-EBV DNA, were correlated with Disease Free Survival (DFS) and Overall Survival (OS) by univariate and multivariate analysis.A total of 130 locally-advanced EBER positive NPC patients were evaluated. Overall, b-EBV DNA was detected in 103 patients (79.2%). Median viral load was 554 copies/mL (range 50-151075), and was positively correlated with T stage (p=0.002), N3a-b vs N0-1-2 stage (p=0.048), type of treatment (ICT followed by CTRT, p=0.006) and locoregional and/or distant disease recurrence (p=0.034). In the overall population, DFS and OS were significantly longer in patients with pre-treatment negative EBV DNA than in positive subjects at the multivariate analysis.Negative b-EBV DNA can be considered as prognostic biomarker of longer DFS and OS in NPC in non-endemic areas. This finding needs confirmation in larger prospective series, with standardized and inter-laboratory harmonized method of plasma EBV DNA quantification.

Human lymphocyte activation gene-3 molecules expressed by activated T cells deliver costimulation signal for dendritic cell activation.

Data have been reported on the in vivo adjuvant role of soluble lymphocyte activation gene-3 (LAG-3) recombinant protein in mouse models and on its ability to support the in vitro generation of human, tumor-specific CTLs. In this study, we show that soluble human rLAG-3 protein (hLAG-3Ig) used in vitro as a single maturation agent induces phenotypic maturation of monocyte-derived dendritic cells and promoted the production of chemokines and TNF-alpha inflammatory cytokine. When given in association with optimal or suboptimal doses of CD40/CD40L, hLAG-3Ig functions as a strong costimulatory factor and induces full functional activation of monocyte-derived dendritic cells that includes the production of high level of IL-12p70. Moreover, evidence is here provided that this costimulatory function licensing dendritic cells to produce IL-12p70 is also a functional property of LAG-3 molecules when expressed in a physiological context by CD4(+) activated T cells. Altogether, these data show for the first time a role of LAG-3 in mediating dendritic cell activation when expressed on the T cell surface or released after specific Ag stimulation in the interspaces of immunological synapses.

Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel, class II HLA-restricted melanoma antigen.

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.