In vitro CAR-T cell killing: validation of the potency assay.

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

Dendritic cell vaccines as cancer treatment: focus on 13 years of manufacturing and quality control experience in advanced therapy medicinal products.

BACKGROUND AIMS: Dendritic cells (DCs) are professional antigen-presenting cells of the mammalian immune system. Ex vivo differentiated DCs represent a unique Advanced Therapy Medicinal Product (ATMP), used in several clinical trials as personalized cancer immunotherapy. The therapy's reliability depends on its capacity to produce high-quality mature DCs (mDCs) in compliance with Good Manufacturing Practices. AIMS: From March 2010 to December 2023, 103 patients were enrolled in multiple clinical trials at the Immuno-Gene Therapy Factory at IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) "Dino Amadori". Six hundred forty-two doses were produced, and the manufacturing process was implemented to optimize production. Our study is a retrospective analysis focusing on the quality control results. METHODS: We retrospectively analyzed the results of the quality control tests carried out on each produced batch, evaluating viability, purity and phenotype of mDCs and their quality in terms of microbiological safety. The data obtained are given with median and interquartile range. RESULTS: The batches were found to be microbiologically safe in terms of sterility, mycoplasma, and endotoxins. An increase in DC maturation markers was found. The release criteria checks showed a high percentage of viability and purity was maintained during the production process. CONCLUSIONS: Our findings have confirmed that the measures implemented have ensured the safety of the products and have contributed to the establishing a robust "Pharmaceutical Quality System." This has enabled many safe mDCs to be produced for clinical trials.

Stability Program in Dendritic Cell Vaccines: A "Real-World" Experience in the Immuno-Gene Therapy Factory of Romagna Cancer Center.

Advanced therapy medical products (ATMPs) are rapidly growing as innovative medicines for the treatment of several diseases. Hence, the role of quality analytical tests to ensure consistent product safety and quality has become highly relevant. Several clinical trials involving dendritic cell (DC)-based vaccines for cancer treatment are ongoing at our institute. The DC-based vaccine is prepared via CD14+ monocyte differentiation. A fresh dose of 10 million DCs is administered to the patient, while the remaining DCs are aliquoted, frozen, and stored in nitrogen vapor for subsequent treatment doses. To evaluate the maintenance of quality parameters and to establish a shelf life of frozen vaccine aliquots, a stability program was developed. Several parameters of the DC final product at 0, 6, 12, 18, and 24 months were evaluated. Our results reveal that after 24 months of storage in nitrogen vapor, the cell viability is in a range between 82% and 99%, the expression of maturation markers remains inside the criteria for batch release, the sterility tests are compliant, and the cell costimulatory capacity unchanged. Thus, the data collected demonstrate that freezing and thawing do not perturb the DC vaccine product maintaining over time its functional and quality characteristics.

Radiotherapy and High-Dose Interleukin-2: Clinical and Immunological Results of a Proof of Principle Study in Metastatic Melanoma and Renal Cell Carcinoma.

High-dose interleukin-2 (HD IL-2) has curative potential in metastatic melanoma (MM) and renal cell carcinoma (RCC). Radiotherapy (RT) kills cancer cells and induces immunomodulatory effects. Prospective trials exploring clinical and immunological properties of combined RT/HD IL-2 are still needed. We designed a phase II, single-arm clinical trial for patients with MM and RCC. The treatment schedule consisted of 3 daily doses of 6-12 Gy of RT to 1-5 non-index metastatic fields, before IL-2 at the first and third treatment cycle. HD IL-2 was administered by continuous infusion for 72 hours and repeated every 3 weeks for up to 4 cycles, thereafter every 4 weeks for a maximum of 2 cycles. The primary endpoint was the immunological efficacy of the combined RT/HD IL-2 treatment (assessed by IFN-γ ELISPOT). Nineteen out of 22 patients were evaluable for immunological and clinical response. Partial response occurred in 3 (15.7%) patients and stable disease was observed in 7 (36.8%). The disease control rate was 52.6% after a median follow up of 39.2 months. According to Common Terminology Criteria for Adverse Events 4.0 (CTCAE 4.0), the majority of toxicities were grade 1-2. Immunological responses were frequent and detected in 16 (84.2%) patients. Increased levels of IL-8 and IL-10 in melanoma, circulating effector memory CD4+ and intratumoral CD8+ T cells in both tumor types were detected after therapy. Overall the treatment was well tolerated and immunologically active. Immunomonitoring and correlative data on tumor and peripheral blood cell subsets suggest that this combination treatment could be a promising strategy for patients progressing after standard treatments.

Potency Assessment of Dendritic Cell Anticancer Vaccine: Validation of the Co-Flow DC Assay.

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC-T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.

Randomized trial comparing fork-tip and side-fenestrated needles for EUS-guided fine-needle biopsy of solid pancreatic lesions.

BACKGROUND AND AIMS: The aim of this study was to compare the performance of EUS-guided fine-needle biopsy using fork-tip or side-fenestrated needles in patients with solid pancreatic lesions. METHODS: A randomized controlled study was conducted in a single academic center on patients who underwent sampling with fork-tip or side-fenestrated 22-gauge or 25-gauge needles. Three passes were performed, each independently evaluated by a blinded pathologist and by endosonographers for macroscopic on-site evaluation (MOSE). The primary outcome was histologic yield; secondary aims were safety, diagnostic yield, sample quality, number of needle passes required to establish a diagnosis, and reliability of MOSE. RESULTS: One hundred ninety-two patients were enrolled. Both 22-gauge and 25-gauge fork-tip needles retrieved significantly higher rates of histologic samples than side-fenestrated needles (P < .013). Safety and diagnostic accuracy were comparable in the 2 arms, whereas sample quality (tissue integrity and blood contamination) was significantly better in the fork-tip group (P < .0001). The median number of diagnostic passes was lower using fork-tip needles (P = .054). The agreement between MOSE and pathologic evaluation was almost perfect in the fork-tip group and fair in the side-fenestrated group. CONCLUSIONS: Both needles showed equivalent safety and diagnostic accuracy. However, fork-tip needles provided a higher rate of extremely good-quality histologic samples and required fewer needle passes to reach a diagnosis. MOSE is a highly reliable tool when fork-tip needles are used compared with side-fenestrated needles. (Clinical trial registration number: NCT03622229.).

Dendritic Cell Vaccination in Metastatic Melanoma Turns "Non-T Cell Inflamed" Into "T-Cell Inflamed" Tumors.

Dendritic cell (DC)-based vaccination effectively induces anti-tumor immunity, although in the majority of cases this does not translate into a durable clinical response. However, DC vaccination is characterized by a robust safety profile, making this treatment a potential candidate for effective combination cancer immunotherapy. To explore this possibility, understanding changes occurring in the tumor microenvironment (TME) upon DC vaccination is required. In this line, quantitative and qualitative changes in tumor-infiltrating T lymphocytes (TILs) induced by vaccination with autologous tumor lysate/homogenate loaded DCs were investigated in a series of 16 patients with metastatic melanoma. Immunohistochemistry for CD4, CD8, Foxp3, Granzyme B (GZMB), PDL1, and HLA class I was performed in tumor biopsies collected before and after DC vaccination. The density of each marker was quantified by automated digital pathology analysis on whole slide images. Co-expression of markers defining functional phenotypes, i.e., Foxp3+ regulatory CD4+ T cells (Treg) and GZMB+ cytotoxic CD8+ T cells, was assessed with sequential immunohistochemistry. A significant increase of CD8+ TILs was found in post-vaccine biopsies of patients who were not previously treated with immune-modulating cytokines or Ipilimumab. Interestingly, along with a maintained tumoral HLA class I expression, after DC vaccination we observed a significant increase of PDL1+ tumor cells, which significantly correlated with intratumoral CD8+ T cell density. This observation might explain the lack of a significant concurrent cytotoxic reactivation of CD8+ T cell, as measured by the numbers of GZMB+ T cells. Altogether these findings indicate that DC vaccination exerts an important role in sustaining or de novo inducing a T cell inflamed TME. However, the strength of the intratumoral T cell activation detected in post-DC therapy lesions is lessened by an occurring phenomenon of adaptive immune resistance, yet the concomitant PDL1 up-regulation. Overall, this study sheds light on DC immunotherapy-induced TME changes, lending the rationale for the design of smarter immune-combination therapies.

Chronic Asymptomatic Pancreatic Hyperenzymemia: A Long-term Follow-up.

OBJECTIVES: Chronic asymptomatic pancreatic hyperenzymemia (CAPH) was described as a benign disease. However, we already described clinically relevant findings requiring surgery or follow-up in half of the subjects. The aim of this study was to evaluate the long-term outcome of CAPH in terms of symptoms and evolution toward chronic pancreatitis. METHODS: Subjects previously enrolled in the first phase of the study (from 2005 to 2010) were reinvestigated from December 2013 to January 2017 with a phone call ± magnetic resonance cholangiopancreatography with secretin stimulation. RESULTS: A total of 133 subjects were eligible for the follow-up study (75 males, 58 females; age, 48.4 [standard deviation {SD}, 14] years); 24 (18%) of them dropped out. During a mean follow-up of 9.3 (SD, 5.2) years after the first diagnosis of CAPH, no episode of acute pancreatitis or abdominal pain was reported. Sixty-three subjects (58%) of 109 underwent magnetic resonance cholangiopancreatography with secretin stimulation with a mean follow-up of 5.7 [SD, 3.1] years (range, 1-11 years). Secretin stimulation-MRCP resulted unchanged in 54 (90%) of 60 subjects, worsened in 3 (5%) and improved in 3 (5%). Two subjects died from causes unrelated to pancreatic disease. CONCLUSIONS: Excluding subjects with a pancreatic disease at index magnetic resonance imaging, CAPH is a benign condition.

Role of SIRT-3, p-mTOR and HIF-1α in Hepatocellular Carcinoma Patients Affected by Metabolic Dysfunctions and in Chronic Treatment with Metformin.

The incidence of hepatocellular carcinoma deriving from metabolic dysfunctions has increased in the last years. Sirtuin- (SIRT-3), phospho-mammalian target of rapamycin (p-mTOR) and hypoxia-inducible factor- (HIF-1α) are involved in metabolism and cancer. However, their role in hepatocellular carcinoma (HCC) metabolism, drug resistance and progression remains unclear. This study aimed to better clarify the biological and clinical function of these markers in HCC patients, in relation to the presence of metabolic alterations, metformin therapy and clinical outcome. A total of 70 HCC patients were enrolled: 48 and 22 of whom were in early stage and advanced stage, respectively. The expression levels of the three markers were assessed by immunohistochemistry and summarized using descriptive statistics. SIRT-3 expression was higher in diabetic than non-diabetic patients, and in metformin-treated than insulin-treated patients. Interestingly, p-mTOR was higher in patients with metabolic syndrome than those with different etiology, and, similar to SIRT-3, in metformin-treated than insulin-treated patients. Moreover, our results describe a slight, albeit not significant, benefit of high SIRT-3 and a significant benefit of high nuclear HIF-1α expression in early-stage patients, whereas high levels of p-mTOR correlated with worse prognosis in advanced-stage patients. Our study highlighted the involvement of SIRT-3 and p-mTOR in metabolic dysfunctions that occur in HCC patients, and suggested SIRT-3 and HIF-1α as predictors of prognosis in early-stage HCC patients, and p-mTOR as target for the treatment of advanced-stage HCC.

Complementary vaccination protocol with dendritic cells pulsed with autologous tumour lysate in patients with resected stage III or IV melanoma: protocol for a phase II randomised trial (ACDC Adjuvant Trial).

INTRODUCTION: Surgery is one of the treatments of choice for patients with a single metastasis from melanoma but is rarely curative. Such patients could potentially benefit from consolidation immunotherapy. Vaccination with dendritic cells (DCs) loaded with tumour antigens elicits a tumour-specific immune response. In our experience, patients who developed delayed type hypersensitivity (DTH) after DC vaccination showed a median overall survival (OS) of 22.9 monthsvs4.8 months for DTH-negative cases. A phase II randomised trial showed an advantage OS of a DC vaccine over a tumour cell-based vaccine (2-year OS 72% vs31%, respectively). Given that there is no standard therapy after surgical resection of single metastases, we planned a study to compare vaccination with DCs pulsed with autologous tumour lysate versus follow-up. METHODS AND ANALYSIS: This is a randomised phase II trial in patients with resected stage III/IV melanoma. Assuming a median relapse-free survival (RFS) of 7.0 months for the standard group and 11.7 months for the experimental arm (HR 0.60), with a two-sided tailed alpha of 0.10, 60 patients per arm must be recruited. An interim futility analysis will be performed at 18 months. The DC vaccine, produced in accordance with Good Manufacturing Practice guidelines, consists of autologous DCs loaded with autologous tumour lysate and injected intradermally near lymph nodes. Vaccine doses will be administered every 4 weeks for six vaccinations and will be followed by 3 million unit /day of interleukin-2 for 5 days. Tumour restaging, blood sampling for immunological biomarkers and DTH testing will be performed every 12 weeks. ETHICS AND DISSEMINATION: The protocol, informed consent and accompanying material given to patients were submitted by the investigator to the Ethics Committee for review. The local Ethics Committee and the Italian Medicines Agency approved the protocol (EudraCT code no.2014-005123-27). Results will be published in a peer-reviewed international scientific journal. TRIAL REGISTRATION NUMBER: 2014-005123-27.

Skewing effect of sulprostone on dendritic cell maturation compared with dinoprostone.

BACKGROUND: Dendritic cells (DCs) are the most efficient antigen-presenting cells and act at the center of the immune system owing to their ability to control both immune tolerance and immunity. In cancer immunotherapy, DCs play a key role in the regulation of the immune response against tumors and can be generated ex vivo with different cytokine cocktails. METHODS: We evaluated the feasibility of dinoprostone (PGE2) replacement with the molecular analog sulprostone, in our good manufacturing practice (GMP) protocol for the generation of DC-based cancer vaccine. We characterized the phenotype and the function of DCs matured in the presence of sulprostone as a potential substitute of dinoprostone in the pro-inflammatory maturation cocktail consisting of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and IL-6. RESULTS: We found that sulprostone invariably reduces the recovery, but does not significantly modify the viability and the purity of DCs. The presence of sulprostone in the maturation cocktail increases the adhesion of single cells and of clusters of DCs to the flask, making them more similar to their immature counterpart in terms of adhesion and spreading proprieties. Moreover, we observed that sulprostone impairs the expression of co-stimulatory molecules and the spontaneous as well as the directed migration capacity of DCs. DISCUSSION: These findings underscore that the synthetic analog sulprostone strongly reduces the functional quality of DCs, thus cannot replace dinoprostone in the maturation cocktail of monocyte-derived DCs.

Metformin and insulin impact on clinical outcome in patients with advanced hepatocellular carcinoma receiving sorafenib: Validation study and biological rationale.

PURPOSE: In 2015, we published a study on a small series of patients with hepatocellular carcinoma (HCC) treated chronically with metformin for type II diabetes mellitus (DM2) who showed a poorer response to sorafenib. The aim of the present study was to validate the prognostic significance of metformin in HCC patients treated with sorafenib, providing a biological rationale for the mechanism of resistance to sorafenib in patients on chronic metformin therapy, and to clarify the role of sirtuin-3 (SIRT-3), a protein involved in metabolic diseases and acknowledged as a tumour suppressor in HCC, in this resistance. PATIENTS AND METHODS: We analysed 279 patients consecutively treated with sorafenib for the clinical analysis. Of the 86 (30%) patients with DM2, 52 (19%) were on chronic treatment with metformin and 34 (12%) with insulin. We included 43 patients with HCC for the biological study: 19 (44.1%) were diabetic and 14 (73.7%) of these received metformin for DM2. SIRT-3 expression was investigated by immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded (FFPE) samples. RESULTS: In HCC patients undergoing chronic treatment with metformin, the use of sorafenib was associated with poor progression-free survival (PFS) and overall survival (OS) (1.9 and 6.6 months, respectively) compared to 3.7 months and 10.8 months, respectively, for patients without DM2 and 8.4 months and 16.6 months, respectively, for patients on insulin (P < .0001). We also observed that SIRT-3 protein expression was significantly higher in patients treated with metformin than in those not taking this medication (65% versus 25%, respectively) (P = .013). CONCLUSIONS: Our findings could be attributed to increased tumour aggressiveness and resistance to sorafenib caused by chronic treatment with metformin.

Dendritic cell vaccination for metastatic melanoma: a 14-year monoinstitutional experience.

Although immunomodulating antibodies are highly effective in metastatic melanoma, their toxicity, related to the activation of T lymphocytes, can be severe. Anticancer vaccines promote a fairly specific response and are very well tolerated, but their effectiveness has yet to be demonstrated. We have been treating patients with advanced melanoma with an autologous dendritic cell vaccine since 2001; to better characterize the safety and efficacy of our product, we designed a retrospective study on all of our patients treated with the vaccine to date. We retrospectively reviewed both case report forms of patients included in clinical trials and medical records of those treated within a compassionate use program. Response was assessed according to the Response Evaluation Criteria In Solid Tumors criteria and toxicity has been graded according to CTCAE 4.0. Although the response rate has been rather low, the median overall survival of 11.4 months and the 1-year survival rate of 46.9% are encouraging, especially considering the fact that data were obtained in a heavily pretreated population and only about one quarter of the patients had received ipilimumab and/or BRAF inhibitors. Multivariate analysis confirmed that the development of an immune response was significantly correlated with a better prognosis (hazard ratio 0.54; P=0.019). The adverse events observed were generally mild and self-limiting. Our analysis confirms the excellent tolerability of our vaccine, making it a potential candidate for combination therapies. As efficacy seems largely restricted to immunoresponsive patients, future strategies should aim to increase the number of these patients.

Interplay Between SIRT-3, Metabolism and Its Tumor Suppressor Role in Hepatocellular Carcinoma.

Sirtuins (SIRT), first described as nicotinamide adenine dinucleotide (NAD+)-dependent type III histone deacetylases, are produced by cells to support in the defense against chronic stress conditions such as metabolic syndromes, neurodegeneration, and cancer. SIRT-3 is one of the most studied members of the mitochondrial sirtuins family. In particular, its involvement in metabolic diseases and its dual role in cancer have been described. In the present review, based on the evidence of SIRT-3 involvement in metabolic dysfunctions, we aimed to provide an insight into the multifaceted role of SIRT-3 in many solid and hematological tumors with a particular focus on hepatocellular carcinoma (HCC). SIRT-3 regulatory effect and involvement in metabolism dysfunctions may have strong implications in HCC development and treatment. Research literature widely reports the relationship between metabolic disorders and HCC development. This evidence suggests a putative bridge role of SIRT-3 between metabolic diseases and HCC. However, further studies are necessary to demonstrate such interconnection.

Transcriptomic analysis identified up-regulation of a solute carrier transporter and UDP glucuronosyltransferases in dogs with aggressive cutaneous mast cell tumours.

Gene expression analyses have been recently used in cancer research to identify genes associated with tumorigenesis and potential prognostic markers or therapeutic targets. In the present study, the transcriptome of dogs that had died because of mast cell tumours (MCTs) was characterised to identify a fingerprint having significant influence on prognosis determination and treatment selection. A dataset (GSE50433) obtained using a commercial canine DNA microarray platform was used. The transcriptome of seven biopsies obtained from dogs with histologically confirmed, surgically removed MCTs, treated with chemotherapy, and dead for MCT-related causes, was compared with the transcriptional portrait of 40 samples obtained from dogs with histologically confirmed, surgically removed MCTs and that were still alive at the end of the follow-up period. Among the differentially expressed genes (DEGs), eight transcripts were validated by quantitative real time PCR and their mRNA levels were measured in a cohort of 22 additional MCTs. Statistical analysis identified 375 DEGs (fold change 2, false discovery rate 5%). The functional annotation analysis indicated that the DEGs were associated with drug metabolism and cell cycle pathways. Particularly, members of solute carrier transporter (SLC) and UDP glucuronosyltransferase (UGT) gene families were identified as dysregulated. Principal component analysis (PCA) of the 22 additional MCTs identified the separate cluster dogs dead for MCT-related causes. SLCs and UGTs have been recently recognised in human cancer as important key factors in tumour progression and chemo-resistance. An in-depth analysis of their roles in aggressive canine MCT is warranted in future studies.

Role of Amylase-α2A Autoantibodies in the Diagnosis of Autoimmune Pancreatitis.

OBJECTIVES: Several serological markers have been reported in autoimmune pancreatitis (AIP) patients. However, only serum IgG4 (sIgG4) is available in clinical practice for AIP diagnosis. Antiamylase α antibodies (AMY-α Abs) have been proposed to diagnose AIP. This study evaluates the utility of AMY-α Abs and sIgG4 for AIP diagnosis. METHODS: Twenty-five AIP patients, 84 disease control groups (31 chronic pancreatitis, 30 acute pancreatitis, 23 pancreatic adenocarcinoma), and 59 healthy donors were prospectively studied. The AMY-α Abs were determined by homemade enzyme-linked immunosorbent assay and sIgG4 by nephelometry. RESULTS: Increased sIgG4 were detected to be present in 52% of AIP, 5% in control groups, and 0% in healthy donors, and AMY-α Abs, respectively, in 76%, 36%, and 2%. sIgG4 was elevated in 92% of the 13 patients with type 1 AIP, but in none of 3 with type 2 and of 8 with not otherwise specified AIP. The AMY-α Abs were present in 79%, 67%, and 75% of type 1, type 2, and not otherwise specified AIP, respectively. Sensitivity and specificity of AMY-α Abs were 76% and 78%, and of sIgG4 50% and 94%. By combining the 2 serological markers, sensitivity was 41%, and specificity was 99%. CONCLUSIONS: The AMY-α Abs may help to diagnosis of AIP and to differentiate AIP subtypes.

Vaccination with autologous dendritic cells loaded with autologous tumor lysate or homogenate combined with immunomodulating radiotherapy and/or preleukapheresis IFN-α in patients with metastatic melanoma: a randomised "proof-of-principle" phase II study.

BACKGROUND: Vaccination with dendritic cells (DC) loaded with tumor antigens elicits tumor-specific immune responses capable of killing cancer cells without inducing meaningful side-effects. Patients with advanced melanoma enrolled onto our phase II clinical studies have been treated with autologous DC loaded with autologous tumor lysate/homogenate matured with a cytokine cocktail, showing a clinical benefit (PR + SD) in 55.5% of evaluable cases to date. The beneficial effects of the vaccine were mainly restricted to patients who developed vaccine-specific immune response after treatment. However, immunological responses were only induced in about two-thirds of patients, and treatments aimed at improving immunological responsiveness to the vaccine are needed. METHODS/DESIGN: This is a phase II, "proof-of-principle", randomized, open-label trial of vaccination with autologous DC loaded with tumor lysate or homogenate in metastatic melanoma patients combined with immunomodulating RT and/or preleukapheresis IFN-α. All patients will receive four bi-weekly doses of the vaccine during the induction phase and monthly doses thereafter for up to a maximum of 14 vaccinations or until confirmed progression. Patients will be randomized to receive:(1.) three daily doses of 8 Gy up to 12 Gy radiotherapy delivered to one non-index metastatic field between vaccine doses 1 and 2 and, optionally, between doses 7 and 8, using IMRT-IMAT techniques;(2.) daily 3 MU subcutaneous IFN-α for 7 days before leukapheresis;(3.) both 1 and 2;(4.) neither 1 nor 2.At least six patients eligible for treatment will be enrolled per arm. Daily 3 MU IL-2 will be administered subcutaneously for 5 days starting from the second day after each vaccine dose. Serial DTH testing and blood sampling to evaluate treatment-induced immune response will be performed. Objective response will be evaluated according to immune-related response criteria (irRC). DISCUSSION: Based upon the emerging role of radiotherapy as an immunologic modifier, we designed a randomized phase II trial adding radiotherapy and/or preleukapheresis IFN-α to our DC vaccine in metastatic melanoma patients. Our aim was to find the best combination of complementary interventions to enhance anti-tumor response induced by DC vaccination, which could ultimately lead to better survival and milder toxicity.

Global gene expression analysis of canine cutaneous mast cell tumor: could molecular profiling be useful for subtype classification and prognostication?

Prognosis and therapeutic management of dogs with cutaneous mast cell tumors (MCTs) depend on clinical stage and histological grade. However, the prognostic value of this latter is still questionable. In the present study, MCT transcriptome was analyzed to identify a set of candidate genes potentially useful for predicting the biological behavior of MCTs. Fifty-one canine MCT biopsies were analyzed. Isolated and purified total RNAs were individually hybridized to the Agilent Canine V2 4x44k DNA microarray. The comparison of reference differentiated and undifferentiated MCT transcriptome revealed a total of 597 differentially expressed genes (147 down-regulated and 450 up-regulated). The functional analysis of this set of genes provided evidence that they were mainly involved in cell cycle, DNA replication, p53 signaling pathway, nucleotide excision repair and pyrimidine metabolism. Class prediction analysis identified 13 transcripts providing the greatest accuracy of class prediction and divided samples into two categories (differentiated and undifferentiated), harboring a different prognosis. The Principal Component Analysis of all samples, made by using the selected 13 markers, confirmed MCT classification. The first three components accounted for 99.924% of the total variance. This molecular classification significantly correlated with survival time (p = 0.0026). Furthermore, among all marker genes, a significant association was found between mRNA expression and MCT-related mortality for FOXM1, GSN, FEN1 and KPNA2 (p<0.05). Finally, marker genes mRNA expression was evaluated in a cohort of 22 independent samples. Data obtained enabled to identify MCT cases with different prognosis. Overall, the molecular characterization of canine MCT transcriptome allowed the identification of a set of 13 transcripts that clearly separated differentiated from undifferentiated MCTs, thus predicting outcome regardless of the histological grade. These results may have clinical relevance and warrant future validation in a prospective study.

DNA and RNA isolation from canine oncologic formalin-fixed, paraffin-embedded tissues for downstream "-omic" analyses: possible or not?

Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective "-omic" investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2-associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.

Low-dose temozolomide before dendritic-cell vaccination reduces (specifically) CD4+CD25++Foxp3+ regulatory T-cells in advanced melanoma patients.

BACKGROUND: In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. METHODS: Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. RESULTS: Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. CONCLUSIONS: Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.