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We reviewed the clinical results of a dendritic cell-based phase II clinical vaccine trial in stage IV melanoma and analyzed a patient subgroup treated with standard therapies after stopping vaccination. From 2003 to 2009, 24 metastatic melanoma patients were treated with mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin and low-dose interleukin-2. Overall response (OR) to vaccination was 37.5% with a clinical benefit of 54.1%. All 14 responders showed delayed type hypersensitivity positivity. Median overall survival (OS) was 15 months (95% CI, 8-33). Eleven patients underwent other treatments (3 surgery, 2 biotherapy, 2 radiotherapy, 2 chemotherapy, and 4 biochemotherapy) after stopping vaccination. Of these, 2 patients had a complete response and 5 a partial response, with an OR of 63.6%.Median OS was 34 months (range 16-61). Our results suggest that therapeutic DC vaccination could favor clinical response in patients after more than one line of therapy. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Glioblastoma (GBM) is a poor prognosis grade 4 glioma. After surgical resection, the standard therapy consists of concurrent radiotherapy (RT) and temozolomide (TMZ) followed by TMZ alone. Our previous data on melanoma patients showed that Dendritic Cell vaccination (DCvax) could increase the amount of intratumoral-activated cytotoxic T lymphocytes., Methods: This is a single-arm, monocentric, phase II trial in two steps according to Simon's design. The trial aims to evaluate progression-free survival (PFS) at three months and the safety of a DCvax integrated with standard therapy in resected GBM patients. DCvax administration begins after completion of RT-CTwith weekly administrations for 4 weeks, then is alternated monthly with TMZ cycles. The primary endpoints are PFS at three months and safety. One of the secondary objectives is to evaluate the immune response both in vitro and in vivo (DTH skin test)., Results: By December 2022, the first pre-planned step of the study was concluded with the enrollment, treatment and follow up of 9 evaluable patients. Two patients had progressed within three months after leukapheresis, but none had experienced DCvax-related G3-4 toxicities Five patients experienced a positive DTH test towards KLH and one of these also towards autologous tumor homogenate. The median PFS from leukapheresis was 11.3 months and 12.2 months from surgery., Conclusions: This combination therapy is well-tolerated, and the two endpoints required for the first step have been achieved. Therefore, the study will proceed to enroll the remaining 19 patients. (Eudract number: 2020-003755-15 https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-003755-15/IT)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ridolfi, Gurrieri, Riva, Bulgarelli, De Rosa, Guidoboni, Fausti, Ranallo, Calpona, Tazzari, Petrini, Granato, Pancisi, Foca, Dall'Agata, Bondi, Amadori, Cortesi, Zani, Ancarani, Gamboni, Polselli, Pasini, Bartolini, Maimone, Arpa and Tosatto.)

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 (r 2  ≥ 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., (© 2024. The Author(s).)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bulgarelli, Piccinini, Petracci, Pancisi, Granato, de Rosa, Guidoboni, Petrini, Ancarani, Foschi, Romeo, Tontini, De Giorgi, Lolli, Gentili, Valmorri, Rossi, Ferroni, Casadei, Cortesi, Crudi and Ridolfi.)

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.

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., (Copyright © 2019 Bulgarelli, Tazzari, Granato, Ridolfi, Maiocchi, de Rosa, Petrini, Pancisi, Gentili, Vergani, Piccinini, Carbonaro, Leone, Foschi, Ancarani, Framarini and Guidoboni.)

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.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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 (PGE 2 ) 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-1β) 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., (Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Purpose: The increasing use of breast-conserving surgery makes it essential to identify biofunctional profiles responsible for the progression of in situ to invasive carcinomas to facilitate the detection of lesions that are most likely to relapse or progress and, thus, to be able to offer patients tailored treatment options. Our objective was to analyse and compare biofunctional profiles in ductal carcinomas in situ (DCIS) and invasive ductal carcinomas (IDC). We also aimed to identify markers in tumor and normal surrounding tissues that may be predictive of locoregional recurrence in patients with DCIS., Methods: Biofunctional parameters including mitotic activity, estrogen receptor, progesterone receptor, microvessel density (MVD), c-kit and p27 expression were evaluated in 829 in situ and invasive carcinomas. The impact of the biomarker profiles of DCIS, IDC and normal surrounding tissues on loco-regional recurrence was analyzed., Results: A progressive increase in cell proliferation and a concomitant decrease in steroid hormone receptor-positive lesions was observed during the transition from in situ to invasive carcinomas, as also within each subgroup as grade increased. Conversely, p27 expression and MVD dramatically decreased during the transition from in situ to invasive carcinomas. Finally, we found that a low c-kit expression was indicative of IDC relapse., Conclusions: Cell proliferation, hormonal and differentiation characteristics differed in DCIS with respect to IDC, and the main variation in the transition between the two histologic lesions was the decrease in p27 expression and MVD.

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.

Dendritic cells (DCs) are unique specialized antigen-presenting cells capable of priming naive T cells and inducing antigen-specific cytotoxic T lymphocytes. This study presents an update of clinical results from a DC-based phase I-II clinical vaccine trial in stage IV melanoma. From 2003 to 2010, 27 patients with metastatic melanoma were treated with mature DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin and with subcutaneous low-dose interleukin-2. Delayed-type hypersensitivity (DTH) tests for in-vivo immunomonitoring were performed at baseline and every four vaccinations thereafter. Two complete, two mixed and six partial responses, and five stable diseases were observed (overall response, 37.0%; clinical benefit, 55.5%). All 15 responders showed DTH positivity. A median overall survival of 22.9 months [95% confidence interval (CI): 13.4-61.3] for DTH-positive patients (19) and 4.8 months (95% CI: 3.9-11.9) for DTH-negative patients (8; log rank=7.26; P=0.007) was observed. The overall median overall survival was 16 months (95% CI: 9-33). Our results would seem to highlight a relationship between positive-DTH test and an improved survival.

Background: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved., Methods: In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100A). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor., Results: We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr., Conclusions: The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

Previous studies have shown that tumor endothelial markers (TEMs 1-9) are up modulated in immunosuppressive, pro-angiogenic dendritic cells (DCs) found in tumor microenvironments. We recently reported that monocyte-derived DCs used for vaccination trials may accumulate high levels of TEM8 gene transcripts. Here, we investigate whether TEM8 expression in DC preparations represents a specific tumor-associated change of potential clinical relevance. TEM8 expression at the mRNA and protein level was evaluated by quantitative real-time RT-PCR and cytofluorimetric analysis in human clinical grade DCs utilized for the therapeutic vaccination of 17 advanced cancer patients (13 melanoma and 4 renal cell carcinoma). The analyses revealed that DCs from patients markedly differ in their ability to up-modulate TEM8. Indeed, mDCs from eight non-progressing patients [median overall survival (OS) = 32 months, all positive to the delayed-type hypersensitivity test (DTH)], had similar TEM8 mRNA expression levels [mDCs vs. immature iDCs; mean fold increase (mfi) = 1.97] to those found in healthy donors (mfi = 2.7). Conversely, mDCs from nine progressing patients (OS < 5 months, all but one with negative DTH) showed an increase in TEM8 mRNA levels (mfi = 12.88, p = 0.0018). The present observations suggest that TEM8 expression levels in DC-based therapeutic vaccines would allow the selection of a subgroup of patients who are most likely to benefit from therapeutic vaccination.

Objectives: This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults., Materials and Methods: Cultures of multicellular spheroids were obtained from human lung tissue specimens after mechanical and enzymatic digestion. Tissue-specific markers were detected on their cells by immunohistochemical and immunofluorescent techniques. Ultrastructural morphology of the spheroids (termed as bronchospheres) was evaluated by electron microscopy, gene expression analysis was performed by reverse transcription-polymerase chain reaction, and gene down-regulation was analysed by an RNA interference technique., Results: Bronchospheres were found to be composed of cells with high expression of stem cell regulatory genes, which was not or was only weakly detectable in original tissues. Morphological analysis showed that bronchospheres were composed of mixed phenotype cells with type II alveolar and Clara cell features, highlighting their airway resident cell origin. In addition to displaying specific pulmonary and epithelial commitment, bronchospheres showed mesenchymal features. Silencing of the Slug gene, known to play a pivotal role in epithelial-mesenchymal transition processes and which was highly expressed in bronchospheres but not in original tissue, led bronchospheres to gain a differentiated bronchial/alveolar phenotype and to lose the stemness gene expression pattern., Conclusions: Ours is the first study to describe ex vivo expansion of stem/progenitor cells resident in human lung epithelia, and our results suggest that the epithelial-mesenchymal transition process, still active in a subset of airway cells, may regulate transit of stem/progenitor cells towards epithelial differentiation.

Non-small cell lung cancer (NSCLC) is the most lethal malignant tumor and is also considered one of the most chemoresistant cancers. Despite the benefits obtained from platinum-based therapy, the majority of patients treated will progress and die. In the continuing quest for personalized therapy based on the biomolecular characteristics of each single patient, clinical practice now seems to be oriented towards combining conventional drugs with molecular-targeted agents. In the present study, we evaluated the antitumor activity of docetaxel, one of the most widely used drugs for second-line treatment, and Iressa, an EGFR-targeting tyrosine kinase inhibitor, administered singly or in sequence. The study was performed on three human NSCLC cell lines (ChaGo-K1, CAEP and RAL) that exhibit different expression of proliferation and apoptosis-related markers, and do not harbor EGFR mutations. The efficacy of docetaxel and Iressa differed in the three cell lines and an important synergistic interaction was observed with the sequence 1-h docetaxel --> 72-h Iressa during which Iressa doubled the fraction of docetaxel-induced apoptotic cells, amplifying a caspase-dependent apoptosis and inhibiting docetaxel-induced p21 hyperexpression. Moreover, the important role of MAPK-dependent modulation of this molecular marker was shown using a specific inhibitor. The results from the present preclinical study demonstrate the cytotoxic activity of Iressa and its ability to increase taxane activity in a model that does not harbor EGFR-specific mutations, thus highlighting the importance of focusing on alternative molecular targets of Iressa activity.

Purpose: Previous studies have shown that telomerase activity in bladder washings and voided urine represents an important noninvasive tool for bladder cancer diagnosis. With the present case-control study conducted on 212 women, including 144 healthy individuals and 68 patients, at first diagnosis of bladder cancer we confirmed previously obtained diagnostic results and improved the accuracy of this diagnostic assay., Materials and Methods: Telomerase activity was evaluated by quantitative telomeric repeat amplification protocol assay and expressed as arbitrary enzymatic units., Results: At the best overall cutoff of 50 arbitrary enzymatic units sensitivity was 87% and specificity was 66%. A breakdown analysis as a function of age showed a higher assay accuracy in women younger than 75 years (sensitivity 91% and specificity 69%) compared to older women (sensitivity 64% and specificity 59%)., Conclusions: Other reasons in addition to age may account for the lower specificity in women with respect to men. In particular, a high number of telomerase positive nonurothelial cells in urine from females could be responsible for false-positive telomeric repeat amplification protocol results. Urine telomerase activity detected by telomeric repeat amplification protocol appears to be a good diagnostic tool in females although it is more accurate in younger than in older women.

Objective: Our work evaluated the potential role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) serum levels with respect to that of conventional serum tumour markers, CEA and CA 15-3, in monitoring the course of metastatic breast cancer in 56 female patients treated with cytotoxic chemotherapy., Methods: VEGF and bFGF concentrations were determined using a quantitative sandwich enzyme immunoassay technique. The positive predictive value (PPV) of each marker and of marker combinations for different types of clinical response was calculated., Results: The highest PPV for overall disease control was shown by bFGF (70%), which also showed the highest PPV for both partial response (36.4%) and stable disease (63.2%). CEA showed the highest predictive value for progression (69.2%). A combined increase in CEA and bFGF or VEGF was associated with disease progression in all patients., Conclusions: Information provided by angiogenic factor levels seems to be independent of and is possibly complementary to that provided by conventional serum markers. bFGF showed the maximum predictive value for disease control and provided additional information to that obtained from CEA or CA 15-3 evaluation. It could therefore be a promising candidate for monitoring response to chemotherapy in advanced breast cancer., (Copyright (c) 2006 S. Karger AG, Basel.)

High expression of the epidermal growth factor receptor (EGFR) family confers a growth advantage on malignant cells in various tumor types. Most pancreatic cancers express EGFR, which seems to play an important role in the acquisition of aggressive clinical behaviour and in tumor invasion. Iressa (ZD1839), a quinazoline tyrosine kinase inhibitor selective for the EGF receptor, has shown good anti-tumor activity in both preclinical and clinical studies. Using two pancreatic cancer cell lines that express different EGFR and ErbB-2 levels, we analyzed the activity of Iressa and evaluated its modulation effect on four conventional cytotoxic drugs: gemcitabine, oxaliplatin, docetaxel and SN38. Iressa was tested at scalar doses up to the plasma peak level concentration and showed a similar weak cytostatic effect in both cell lines. Conversely, an additive or weak synergistic effect was observed when the drug was administered simultaneously with or following cytotoxic drugs. Our data show that Iressa has only a weak activity at doses within the plasmatic peak concentration and that its effect is independent of EGFR and p42/p44 expression and phosphorylation levels. This is in agreement with recent literature data that attribute an essential role to a specific EGFR mutation in mediating response to Iressa. This mutation was absent in both pancreatic cell lines tested.

Introduction: The aim of the present study was to analyze the relationship between the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in breast cancer cells and the corresponding serum levels in individual patients. The study also evaluated the potential of serum levels of the two growth factors as diagnostic markers in a case-control study., Methods: VEGF expression and bFGF expression were determined in 62 and 63 tumor samples, respectively. Serum VEGF and bFGF levels were determined in 54 and 65 healthy women and in 69 and 73 breast cancer patients, respectively, using a quantitative sandwich enzyme immunoassay technique., Results: A direct correlation was observed between VEGF expression and bFGF expression in individual tumors (P = 0.001) and between serum levels (P = 0.038) in individual patients, but not between tumor cell expression and the corresponding serum level for either growth factor. Median values of serum levels in healthy women and breast cancer patients were not different for VEGF (P = 0.055), but were significantly different for bFGF (P < 0.001). The receiver operating characteristic curve identified a serum bFGF concentration of 1.0 pg/ml, with 84.9% sensitivity and 63.1% specificity, as the best cut-off value to discriminate between healthy women and breast cancer patients. An age-based subgroup analysis showed that serum values of patients older than 70 years of age mainly contributed to the high accuracy., Conclusions: Our data repropose bFGF as a noninvasive diagnostic tool for breast cancer.

Alterations in the expression of signal activation molecules, such as the T-cell receptor (TCR) zeta and epsilon chains and p56lck tyrosine kinase, are described in tumor-infiltrating lymphocytes (TIL). The aim of this study was to ascertain if such molecules were present in near-tumor-tissue lymphocytes (NTTL) and peripheral blood lymphocytes (PBL), as well as TIL, of renal cell carcinoma patients, to verify whether this tumor induces immunosuppression only locally or affects distant lymphocytes as well. Tissue from the tumor and from healthy nearby sites, as well as blood samples, were obtained from 27 consecutive patients who had undergone radical nephrectomy for renal cell carcinoma. Phenotype analysis and immunohistochemical staining of the TCR zeta and epsilon chains and p56lck were performed with standard techniques on TIL, NTTL, and PBL, and values were compared for each patient. Low expression of the TCR zeta chain and an almost complete absence of TCR epsilon chain and p56lck expression was observed in TIL (median values: 10% for zeta chain and 0% for epsilon and p56lck). In NTTL, these signal transduction molecules were expressed by a higher percentage of cells (60%, 50%, and 60%, respectively; p=0.000 vs. TIL), whereas PBL showed an almost normal expression of zeta and epsilon chains (80% and 90%, respectively; p=0.000 vs. TIL). Conversely, p56lck was detected in a greater proportion of NTTL than PBL (50% vs. 10%; p=0.001). The absence or the very low expression of signaling activation molecules in TIL compared with NTTL and PBL in renal cancer patients suggest that tumor-induced immunosuppression generally occurs or starts locally.

Purpose: We analyzed the clinical relevance of HER-2 expression, widely investigated in breast cancer but with contradictory results, in the largest case series of node-negative breast cancer patients investigated to date., Patients and Methods: The pure prognostic value of HER-2 expression was investigated in 529 patients treated with locoregional therapy alone until early relapse. Proliferative activity was evaluated as [3H]thymidine labeling index and HER-2 expression by immunohistochemistry. All biologic determinations were conducted within the context of an intra- and interlaboratory National Quality Control Program., Results: HER-2 expression was not related to relapse-free survival in the overall series but was a significant discriminant of prognosis in the subgroup of patients with rapidly proliferating tumors. Six-year rate of relapse was 40% for patients with highly (> or =30%) positive tumors and 26% for those with weakly HER-2-expressing tumors (P =.039)., Conclusion: HER-2 expression in association with proliferative activity identifies a subgroup of node-negative breast cancer patients with the worst prognosis, who are candidates for specific intensive adjuvant therapy.

We investigated the effects of interleukin-2 (IL-2) exposure on T-cell signal transduction molecules and apoptosis markers in tumour-infiltrating lymphocytes (TIL) isolated from 20 melanoma and 16 colorectal carcinoma metastases and expanded in vitro for therapeutic reinfusion. Before IL-2 culture, TIL showed undetectable or very low levels of T-cell receptor (TCR) epsilon chain, p56(lck), Fas ligand (FasL) and Bax expression, while Bcl-2 values were elevated. Cancer cells were characterised by low or absent Fas and Bcl-2 and high Bax expression. Notably, they also expressed FasL. After 41-48 days of IL-2 culture, TCR epsilon chain and p56(lck) expression of TIL rose to median values of approximately 80 and 30% positive cells, respectively (P<0.001), FasL expression was detected in 45% cells from melanomas (P<0.001) and in 3% from colorectal carcinomas (P=0.09), and Bax-positive cells increased from 17.5 to 70% (P=0.005). Moreover, TCR zeta chain-positive cells were significantly increased from baseline (P=0.001), Bcl-2-positive cells dropped from 50 to 1% (P=0.007) and perforin content was high, while Fas expression was not significantly modified by IL-2 culture. In conclusion, our data suggest that the degree of immunosuppression in TIL from melanomas and colorectal carcinomas is very high, and the apoptosis markers' repertoire of cancer cells resembles that of immune-privileged tissue. Interleukin-2 culture appears to restore lymphocyte activation mechanisms, resulting in consistent FasL expression and perforin production.

We investigated a number of biological markers, evaluated under strict intralaboratory quality control conditions, in terms of their role in predicting clinical outcome of patients with colon cancer treated with 5-FU-containing regimens. Colon cancer tissue from 263 patients enrolled onto two randomised clinical trials were studied for their cytofluorimetrically determined DNA content and their immunohistochemically evaluated microvessel density, vascular endothelial growth factor expression, thymidylate synthase expression and tumour lymphocyte infiltration. Disease-free survival and overall survival of patients were analysed as a function of the different variables. At a median follow up of 57 months, age, gender and Dukes' stage showed an impact on disease-free survival, whereas no biological marker emerged as an indicator of better or worse disease-free survival. Only histological grade and Dukes' stage were found to influence overall survival. The different biological variables, studied with particular attention for determination reliability, proved to have no impact on the clinical outcome of patients with colon cancer. Therefore, other markers must be identified to complement clinico-pathological variables in the management of this disease., (Copyright 2002 Cancer Research UK)

Vascular endothelial growth factor (VEGF) has emerged as one of the most important angiogenic growth factors from experimental in vitro and in vivo studies. In the present study, we investigated the relationship between VEGF expression and microvessel density (MVD) and defined their prognostic relevance on a series of 242 patients with node-negative breast cancer, using immunohistochemical methods. In parallel, estrogen and progesterone receptors were quantitatively assessed using the dextran-charcoal technique and cell proliferation was evaluated as S-phase cell fraction according to (3)H-thymidine-labeling index (TLI). The percentage of VEGF-expressing cells varied from 0-95% in the different tumors and was unrelated to menopausal status, tumor size or steroid receptor status. Conversely, a significant inverse relation was observed with patient age or tumor cell proliferation, albeit with very poor correlation coefficients. A significant relation was observed between VEGF expression and MVD (r(s) = 0.55, p < 0.001). Clinical outcome analyzed as a function of high and low VEGF expression showed slight differences in terms of both disease-free survival (DFS) and overall survival (OS) that never reached statistical significance. Moreover, the trend was paradoxically in favor of patients with highly VEGF-expressing tumors. Finally, DFS and OS curves, when analyzed as a function of VEGF expression or MVD, were superimposable. In conclusion, our study did not highlight a prognostic relevance of VEGF expression in patients with node-negative breast cancer, as already observed for MVD., (Copyright 2001 Wiley-Liss, Inc.)

The development of cancer in the breast and in other sites is a complex process requiring a number of different genetic and epigenetic alterations. The accumulation of the genetic changes is thought to underlie the progression from precancerous lesions to carcinomas. The expression of p27/kip1 protein, a cyclin-dependent kinase inhibitor, was investigated by immunohistochemistry in normal epithelial specimens, benign alterations, and malignant lesions of the breast. The number of p27/kip1-positive cells ranged from none to more than 98% in the overall series. Wide ranges of p27/kip1-positive cells were consistently observed within each histological category, but the median value progressively decreased in typical hyperplasia and fibroadenoma, with an even more marked reduction in malignant lesions, compared with normal epithelium. Moreover, the percentage of cells expressing p27/kip1 in tumours was about three times lower in invasive than in in situ lesions and was inversely related to tumour size, but not to lymph node involvement. In conclusion, the degree to which p27 expression is altered in typical hyperplastic lesions and fibroadenomas indicates that the deregulation of p27 may occur very early on during breast cell transformation, but the usefulness of its determination to categorize subgroups of lesions at different risk of evolution remains somewhat doubtful., (Copyright 2001 John Wiley & Sons, Ltd.)

The predictivity of tumour size, oestrogen (ER) and progesterone (PgR) receptors, 3H-thymidine labelling index (TLI), c-erbB-2 and p27kip1 expression on clinical outcome was analysed on a consecutive series of 118 postmenopausal patients with ER-positive, node-positive tumours. All patients were treated with surgery +/- radiotherapy and adjuvant tamoxifen (30 mg/day) for at least 2 years. TLI, ER, c-erbB-2 and p27kip1 were generally unrelated to each other. PgR was directly related to ER and inversely to c-erbB-2. Tumour size was inversely related to both c-erbB-2 and p27kip1 expression. At a median follow-up of 75 months, 5-year relapse-free survival was significantly lower for patients with very rapidly proliferating (HR = 2.61, 95% CI = 1.34-5.08), PgR negative (HR = 2.76, 95% CI = 1.43-5.33) or relatively low ER content (HR = 2.20, 95% CI = 1.14-4.25) tumours than for patients with tumours expressing the opposite biological profiles. Overall survival was also significantly different as a function of TLI (HR = 3.47, 95% CI = 1.52-7.93) and PgR (HR = 2.27, 95% CI = 1.00-5.15). TLI and PgR maintained an independent relevance in multivariate analysis and together were capable of identifying subgroups of patients at significantly different risk of relapse and death.

It is generally thought that future advances in the treatment and cure of breast cancer patients will be made possible through a deeper understanding of tumor biology and an improved capability to define the prognosis of each single patient. This will lead to the formulation of new, more selective, and patient-tailored therapies. It is therefore important, when studying potential prognostic factors, to follow methodologic requirements and guidelines which involve the carrying out of prospective studies as confirmatory steps. Repeatedly or recently investigated prognostic markers (tumor size, menopausal status, ER, PgR, 3H thymidine labeling index, c-erbB-2 and p27 expression) were evaluated on a series of 286 prospectively recruited node negative breast cancer patients who underwent loco-regional treatment alone and were closely followed. The individual and relative prognostic contribution of each variable with respect to other factors, as well as their ability to identify node negative patients at risk, were assessed by univariate and multivariate analysis. At a five-year follow-up, only tumor size (p = 0.021) and TLI (p = 0.016) individually proved to be significant prognostic indicators of relapse-free survival. Conversely, p27 expression was not related to RFS and c-erbB-2 expression appeared to have only a short-term effect on patient prognosis. TLI and tumor size, tested in multivariate analysis along with ER and menopausal status, maintained their independent prognostic relevance. The study, performed on a large series of node-negative patients given loco-regional treatment alone, for the first time prospectively recruited, showed the prognostic relevance of TLI and its independence from other clinico-pathologic and biologic factors over a five-year period.