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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.

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.

IntroductionSurgery 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 analysisThis 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 disseminationThe 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 number2014-005123-27.

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 and Objectives Conventional chemotherapy has not proven effective in improving long-term results of surgery for liver metastases from colorectal cancer. We assessed the usefulness of immunotherapy with tumor infiltrating lymphocytes (TIL) plus Interleukin-2 (IL-2) as adjuvant treatment. Methods Between 1995 and 1998, 47 patients were enrolled onto a prospective protocol; 25 entered the treatment group (A) and 22 entered the control group (B). All patients had undergone radical liver resection. TIL obtained from surgical specimens from group A patients were cultured and activated in vitro with IL-2, then reinfused into the patients with IL-2. We investigated pre- and post-IL-2 stimulation expression of T cell receptor (TCR) ζ- and e-chains, p56lck, Fas, and Fas-L by TIL immunostaining. Results Fourteen patients from group A (56%) received immunotherapy; 14 from group B (60%) underwent conventional chemotherapy, and the remaining 19 patients did not receive any treatment. No significant differences between the two groups were found in the actuarial and disease-free survival (DSF) rates after 1, 3, and 5 years. After IL-2 exposure, TCR ζ-chain expression significantly increased (P = 0.001); An increase in TCR e-chain expression (P = 0.04), and p56lck (P = 0.03) was detected; TCR e-chain expression was significantly increased in disease-free patients compared to those who relapsed (P = 0.04). Fas-L expression was correlated with the TCR e-chain and p56lck levels (P = 0.05). Conclusions Our data suggest that we are still a long way from being able to propose TIL + IL-2 treatment as an effective adjuvant therapy. However, the results confirm that the biological indicators examined could play an important role in modulating immunitary response against tumor cells. J. Surg. Oncol. 2004;87:46–52. © 2004 Wiley-Liss, Inc.

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.

The most recent advances in immunology bear witness to the fact that tumors, in particular melanoma, escape recognition by the host's immune system and can locally inactivate its effectors, T-cells and antigen presenting cells. There is, however, preclinical evidence that the immune system, opportunely stimulated, is capable of recognizing and killing tumor cells. It has been verified that the activation of autologous dendritic cells, derived from peripheral blood and pulsed with tumor antigens, results in the specific stimulation of T-cells against the tumor. Preliminary data from dendritic cell vaccination trials, mainly of advanced melanoma, show unequivocal evidence of immunization and of the first clinical responses. Many questions remain to be answered before more effective and widespread use of this type of vaccination is possible, especially in the early stages of the disease.

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.

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.

Cell kinetics holds a prominent role among biological factors in predicting clinical outcome and response to treatment in neoplastic patients. Different cell kinetic variables are often considered as valid alternatives to each other, but the limited size of case series analysed in several studies and the lack of simultaneous determinations of all the variables on the same tumours do not justify this conclusion. In the present study, the correlation between [3H]thymidine labelling index ([3H]dT LI), flow cytometric S phase cell fraction (FCM-S) and Ki-67 immunoreactivity (Ki-67/MIB-1) was verified and the type of correlation with the most important clinical, pathological and biological patient and tumour characteristics was investigated in a very large series of breast cancer patients. Ki-67/MIB-1, FCM-S and [3H]dT LI were determined in 609, 526 and 485 patients, respectively, and all three cell proliferation indices were evaluated in parallel on the same tumour in a series of 330 breast cancer patients. All the cell kinetic determinations were performed within the context of National Quality Control Programmes. Very poor correlation coefficients (ranging from 0.37 to 0.18) were observed between the different cell kinetic variables determined in parallel on the same series of breast cancers. Moreover, Ki-67/MIB-1 and FCM-S showed a significant relationship with histological type, grade and tumour size, whereas statistically significant correlations were not observed for [3H]dT LI. In conclusion, the results show that the different cell kinetic variables provide different biological information and cannot be considered as alternatives to each other.

Microvessel density (MVD) of breast cancer is widely regarded as a prognostic factor, but results from studies on the most important case series have produced conflicting results. The present study was performed with confirmatory intent to define the prognostic relevance of MVD on a series of 378 node-negative-breast-cancer patients, much larger than any other series previously analyzed. Microvessels were stained using Factor-VIII antibody and an immunoperoxidase reaction. MVD was determined independently by 2 observers according to Weidner's methods. In parallel, cell proliferation was evaluated as S-phase fraction and determined according to the 3H-thymidine-labeling index method (TLI). Estrogen and progesterone receptors were quantitatively assessed using the dextran-charcoal technique. Tumor MVD varied greatly from tumor to tumor (2 to 232 MV/mm2) and was unrelated to patient age and menopausal status, or to tumor size, histology and steroid-receptor status. A significant (p = 0.004) but weak inverse correlation (rs = -0.188) was observed with cell proliferation. Univariate analysis using 40 MV/mm2 as cut-off showed an inverse relation with 5-year relapse-free survival (82% vs. 71%, p = 0.018). This finding was limited to very small tumors, slowly proliferating tumors and ER-negative tumors. Multivariate analysis identified tumor size and TLI, but not MVD, menopausal status or ER as independent prognostic factors.

Aims and background Adoptive immunotherapy with tumor infiltrating lymphocyte (TIL) reinfusion plus continuous interleukin-2 (IL-2) infusion could represent an innovative way of treating immunogenic tumors. This study therefore recruited melanoma, colorectal and renal carcinoma patients whose metastases had been surgically removed. Study design The treatment was initially given to 22 patients with advanced disease and more recently to 39 disease-free (DF) patients after radical metastasectomy. The latter group was selected in view of a theoretically better lymphocyte/tumor cell ratio and with the aim to improve disease-free and overall survival (DFS-OS) in very high risk patients. The starting IL-2 dose was 12 MlU/day (West's schedule); doses were modulated on the bases of toxicity parameters. Even though patients received different total amounts of IL-2, all of them completed the treatment. Results The treatment was offered to 22 advanced-stage cancer patients (12 melanomas, 9 colorectal carcinomas, 1 kidney carcinoma). Few and short stabilizations were observed with a median survival of 12 months (range, 3-29). Subsequently, another 39 patients were treated in an adjuvant setting after radical metastasectomy (18 melanomas, 19 colorectal carcinomas, 2 kidney cancers). Eleven out of 17 DF melanoma patients (64.7%) are still free of disease after a median of 37+ months (range, 5+ - 69+). In the group of DF colorectal cancer patients eight (44.4%) are still DF after a median of 21+ months (range, 7+ - 67+ months). One of the two patients with kidney cancer is still DF after 28+ months. Two patients (1 melanoma and 1 colorectal cancer) had just been treated and were therefore not evaluable. Severe toxicity occurred in three cases but was rapidly resolved. There was a great diversity in IL-2 doses administered; comparison of the total IL-2 dose administered between the patients who are still DF and those who progressed revealed no difference between the two groups of colorectal cancer patients, whereas melanoma patients who progressed rebeived an average IL-2 dose of 6.5 MlU/day versus 15.8 MlU/day in DF patients. No differences were observed in any of the groups between the number of TILs reinfused and clinical response. Conclusions The study is still ongoing; it has been decided to focus on DF melanoma patients after radical metastasectomy, for whom the data seem to be encouraging. Further endpoints of the study are the role of IL-2 dosage in the adjuvant setting, and the possibility to make correlations between biological parameters and clinical results.

Adoptive tumour infiltrating lymphocytes (TIL) in combination with a modulated dosage of interleukin-2 (IL-2) can be used with acceptable toxicity in the treatment of immunogenic tumours. Following an experience of reinfusion in advanced melanoma, colorectal and renal cancer patients, treatment was given to disease-free patients after metastasectomy. The high risk of relapse and favourable ratio between reinfused TIL and possible microscopic residual disease determined this choice of adjuvant treatment. A group of 12 patients with advanced disease (7 melanoma, 4 colorectal carcinoma, 1 kidney carcinoma) were treated with TIL (median 5.8 x 10(10) cells) and IL-2 (West's schedule) modulated towards a lower dosage (from 12 to 6 MIU/day) in order to maintain an acceptable level of toxicity. As treatment was well tolerated, it was offered to another 22 patients in an adjuvant setting after metastasectomy (11 melanoma, 10 colorectal carcinoma, 1 renal cancer), the median dose of TIL reinfused being 4.95 x 10(10) cells. No objective response was observed in advanced patients: all patients progressed after a median of 1.5 months (0-8 months) and median survival was 8 months (3-22+ months). Thirteen patients from the second group are still disease-free after a median of 23+ months (9+ - 47+ months). The remaining 9 patients relapsed after a median of 5 months (3-18 months). Toxicity was moderate as clinical and hepatic/renal function parameters were used to assess the need for dose reductions. Consequently, there was great diversity in IL-2 dosages administered. In particular, there seemed to be a difference in IL-2 doses administered between disease-free cases and those who progressed (17.5 MIU/day versus 7 MIU/day in melanoma patients; 11.2 MIU/day versus 7.1 MIU/day in colorectal cancer patients). By contrast, no differences were observed between number of TIL reinfused and clinical response. Phenotypical characteristics of reinfused TIL were similar to those reported in the literature: 97% were CD3 and 92% were CD8. Aspecific cytolytic activity was evaluated on 12 cases whereas, in 2 melanoma cases, autologous tumour tissue was available for the specific cytotoxicity test. Perforin levels in TIL measured at the end of culture were generally high or very high. Cytokine levels were measured on the supernatant at the end of culture, with an estreme variability in results. Finally, delta chain and p56lck were histologically assessed on the resected tissue from which TIL were cultivated. There were virtually none of the former and a complete absence of the latter, which concurs with data reported in the literature. The same immunocytochemical analysis was carried out on TIL at the end of culture. This time an almost complete restoration of both functions was seen, especially in melanoma patients, who are still free from disease. The study is on-going and it has been decided to focus on disease-free patients after metastasectomy in order to increase the number and possibility of clinical and histological correlations.

Cancer anorexia-cachexia syndrome (CACS), which is characterized by progressive weight loss (WL) and anorexia (A), is present in 50% of advanced cancer patients and in 80% of terminally ill cancer patients. One of the most controversial aspects of CACS is its oetiopathogenesis; experimental studies have identified certain cytokines [Tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), interleukin 6 (IL-6), and gamma interferon (gamma-IFN)] as possible co-factors in the onset of the syndrome. The aim of our study was to investigate the correlation between serum levels of circulating cytokines and severity of CACS. The following series of parameters was identified in 61 patients with advanced and terminal cancer: stage of disease; Karnofsky performance status (KPS) and clinical symptoms; biohumoral, anthropometric and immunological situation; level of circulating cytokines. All these parameters were evaluated for a possible link with WL/A. Our data do not show any significant correlation between circulating cytokines and WL/A. A direct correlation was identified between WL/A and nausea (P = 0.03 and P0.001, respectively) whereas inverse correlations were observed for both factors as regards arm circumference (P0.001 for both), wrist circumference (P0.001 for both), KPS (P0.001 and P = 0.003, respectively) and creatinine (P = 0.005 and P = 0.03, respectively). Other biochemical factors, such as haemoglobin, haematocrit, glycaemia, prealbumin, sodium and chlorine were also correlated with at least one of two clinical parameters in question. Unexpected results were seen in the increases in CD20 and CD4 and in the CD4/CD8 ratio. Serum levels of these cytokines do not, therefore, appear to be critical in the onset of CACS. On the contrary, our findings confirmed the clinico-laboratory picture that is characteristic of CACS. If we consider the possibility that CACS is provoked by an aspecific response of the host's defence mechanisms against prolonged neoplastic attack, the increase in CD4 (helper lymphocytes) could be linked to the persistent response.

3040 Background: Vaccination with dendritic cells (DC) is still a valid experimental option for metastatic melanoma (MM). However, only a few patients experience long-lasting objective responses and the majority of clinical responders afterwards relapse and die. Which mechanisms are actually responsible for this “secondary resistance” to whole tumor antigens-loaded DC vaccines is largely unknown. It has been hypothesized that suppressive immune cell subpopulations, regulatory T cells in particular, may progressively accumulate in tumor tissues thus hampering therapy-induced antitumor immune responses along time. To elucidate this issue we evaluated changes induced by immunologically effective DC vaccination in the composition of tumor-associated T cell subpopulations. Methods: 12 patients with MM previously enrolled in a phase I/II DC vaccine trial and for which tumor tissue taken before and after at least 4 induction vaccine doses were available, were included in the study. Intratumoral lymphocytes were evaluated by CD3, CD4, CD8, FoxP3 and GrB immunostainings, and quantified by a computer-assisted method. A nonparametric two-tailed Wilcoxon signed-rank test was utilized for evaluating differences in the distribution of the number of cell positive for each marker on the total cell counts in pre- and post-vaccine biopsies. Results: Our data showed a considerable and statistically significant decrease of intratumoral FoxP3+regulatory T cells in melanoma tissues after DC vaccination. In addition, the concurrent increase of intratumoral activated cytotoxic T lymphocytes, as shown by CD8 and Granzyme B stainings, indicated that this decrease has likely a functional relevance. Conclusions: Our findings that vaccination with DC loaded with autologous tumor lysate strongly reduces the intratumoral content of regulatory T cells add strength to the rationale for the development of potentially more effective combination schedules where whole tumor antigen-loaded DC vaccine prime and partially activate tumor-specific low-affinity T cells in a first tumor antigen-focusing step, followed by boosting with non-maximal doses of anti-CTLA-4 antibodies.

A 37-year-old patient with liver metastases from gastric cancer was treated with a double adoptive immunotherapy regimen comprising tumor-infiltrating lymphocytes plus interleukin-2 and subsequently local-regional lymphokine-activated killer cells plus interleukin-2 because of an extremely high in vitro cytotoxic specific activity on established gastric cancer cell lines. The necrosis verified in the center of the hepatic metastasis would appear to demonstrate treatment efficacy, but no clinical response was seen. In vitro cytotoxicity data alone are insufficient to predict the clinical efficacy of adoptive immunotherapy.

Natural history of renal cell carcinoma includes metastases to the pancreas. The literature reports that selected patients may have benefits by pancreatic resection in terms of long term survival. We report patient outcome and considerations on immunotherapy approach.From 2001 to 2010 eight patients underwent pancreatic resection for metastases from renal cancer. We reviewed surgical outcome and following treatment (conventional chemotherapy: 5FU-Vindesine; Immunotherapy: Interleukin 2 - Interferon - Dendritic cells) of these patients.All patients underwent radical pancreatic resection (7 spleno-pancreatectomies; 1 segmental pancreatic resection) and were R0 after surgery. No postoperative mortality was reported. Morbidity was 37% (2 distal leakage; 1 pneumonitis). Two patients did not receive any further treatment; 2 patients received conventional chemotherapy; 2 patients received immunotherapy (interleukin2 + interferon); 2 patients received dendritic cells (DC) interleukin-2 infusion. Three years overall survival rate was 55%. Disease free survival after 3 years was 30%.Our data confirm that pancreatic resection should be offered to selected patients with no mortality and low morbidity. Long-term survival is achievable, but recurrence rate after surgery is high. Immunotherapy could be effective to control tumour progression especially in selected cases where DC may be used.

The expression of cathepsin D was evaluated by immunohistochemistry on histologic sections from formalin-fixed samples in a series of 436 primary breast cancers. The fraction of cathepsin D-positive tumor cells was not related to tumor size or hormone receptor status, and only weakly related to proliferative activity, evaluated as the 3H-thymidine labeling index. Conversely, a higher fraction of positive cells was observed in node-positive than in node-negative tumors (p = 0.05). A matched comparison between immunohistochemical and immunoradiometric results on individual tumors was carried out on 100 cases and showed a significant association but with a correlation coefficient of 0.46. The agreement of results from the two assays was higher in ER- than in ER+ tumors, which sometimes showed an immunostaining limited to macrophages and normal epithelial cells. In situ evaluation has the main advantage of being specifically applicable to detection in tumor cells and allows the simultaneous determination of different biologic aspects for a more complete understanding of breast cancer biology.

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.

Biological response modifiers (BRMs) have greatly modified the immunotherapy of tumors. Interleukin-2 (IL-2) has brought about metastasis regression in some cases of malignant tumors, however, when given systemically, it results in high toxicity. More recently, the subcutaneous administration of IL-2 (combined with alpha-interferon, alpha-IFN) seems to be capable of offering the same chances of therapeutic response, but this time with a lower level of toxicity. The Authors report an evaluation of toxicity in 22 patients treated with a combination of IL-2 + alpha-IFN i.m. with or without chemotherapy. The side-effects present in the majority of cases were: fever, diarrhea and asthenia. Approximately 50% of the patients had nausea/vomiting, mucositis, skin rashes, and slight leukopenia. The following side-effects were noted to a much lesser degree, thrombocytopenia, alterations in hepatic and dizziness and cystitis. Only one patient reached 4th degree toxicity, with mucositis, asthenia and skin rash. All the other patients received the treatment without suspensions for toxicity. Biological evaluations will enable us to determine in the future, the cases which can benefit from therapeutic intensification and thus it would seem opportune at this time to use therapy with acceptable toxicity.

Mitogenic properties have been demonstratedin vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p

The action of high-dose medroxyprogesterone acetate (MPA) was studied by analysing the behaviour of colony-forming-unit granulocyte-macrophage (CFU-GM) during chemotherapy. 21 non-pretreated men with locally advanced carcinoma of the head and neck were randomised into two arms: A (11 patients) received three alternating cycles of cisplatin, 5-fluorouracil (CF)/cisplatin, methotrexate, bleomycin, vincristine and then CF every 4 weeks and B (10 patients) were treated with the same schedule plus 1000 mg per day of MPA. MPA was administered 14 days before the start of chemotherapy (day 0) and continued daily up to the 90th day. Bone marrow was harvested in arm A on days 0, +14 and +90, and in B, also on day -14. There was diverse CFU-GM behaviour in the two arms on the 14th day. These data support the hypothesis that the myeloprotective effect of MPA is due to induction of a mitotic rest in the stem cells, which protects them from drug action.

Proliferative activity (expressed as 3H-thymidine labeling index, 3H-TdR LI) was evaluated on a series of 281 primary tumors recruited in two years in 6 different institutions from central Italy. 3H-TdR LI proved to be low in intraductal, or well and moderately differentiated, or hormone receptor positive tumors. Conversely, no relation was observed between 3H-TdR LI and menopause, tumor size, or lymph node involvement. An inverse relation was observed between 3H-TdR LI and hormone receptor content. Specific patterns of 3H-TdR LI value and ER content association were observed as a function of menopause, lymph nodal status, and degree of lymph nodal involvement.

Towards the end of the 1990s, the two opposing theories on immunosurveillance and immunostimulation were extensively studied by researchers in an attempt to understand the complex mechanisms that regulate the relation between tumors and the host's immune system. Both theories probably have elements that would help us to comprehend how the host can induce anti-tumor clinical responses through stimulation of the immune system and which could also give us a deeper insight into the mechanisms of tumor immunosuppression. The model that most resembles the behavior of tumor cells in terms of growth, infiltration and suppression of the immune system of the environment in which they live is undoubtedly that of the embryonic cell. The fetus behaves like an allogenic transplant within the mother's body, using every means it has to escape from and defend itself against the mother's immune system. The majority of these mechanisms are the same as those found in tumor cells: antigenic loss, lack of expression of classic HLA-I molecules, production of immunosuppressive cytokines, induction of lack of expression of co-stimulatory molecules in antigen presenting cells, and induction of apoptosis in infiltrating lymphocytes, with activation of a type Th2 regulatory lymphocyte response. A careful and comparative study of key mechanisms capable of triggering tolerance or cytotoxicity in both embryonic and tumor cells could prove immensely valuable in designing new strategies for anti-tumor immunotherapy.

e14007 Background: Ashigh-dose Interleukin-2 (HDIL2) is a necessary component of a curative treatment strategy in melanoma and renal cell carcinoma (RCC), it is imperative to improve its therapeuti...

Background We present our experience of therapeutic vaccination using dendritic cells (DC) pulsed with autologous tumor antigens in patients with advanced melanoma. Methods Twenty-one pretreated advanced melanoma patients were vaccinated with autologous DC pulsed with 100 μg/ml of autologous-tumor-lysate (ATL) or – homogenate (ATH) and 50 μg/ml of keyhole limpet hemocyanin (KLH). The first 8 patients were treated subcutaneously or intradermally with immature-DC (iDC) (range 4.5 – 82 × 106) and the remaining 13 intradermally with in vitro matured DC (mDC) (range 1.2–26 × 106). Subcutaneous interleukin-2 (3 × 106 IU) was administered from days 3 to 7 of each treatment cycle. Results Three of the 8 iDC patients obtained stabilizations (SD), each of 6 months' duration. The 13 mDC patients showed 1 complete response (8 months), 1 partial response (3 months), 2 mixed responses (6 and 12 months) and 3 SD (9, 7+, and 3+ months). Overall responses (OR) were observed in 4/21 (19%) patients, or 4/13 (30.7%) considering mDC treatment only. 10/21 (47.6%) patients showed non progressive disease (NPD), with 7/13 (53.8%) cases of NPD for mDC-treated patients. No major toxicities were observed. The positive delayed-type hypersensitivity (DTH) test to ATL/ATH and/or KLH correlated with increased overall survival (OS). Median OS was 24 months (range 3 – 45) for the 10 DTH-positive (1 iDC and 9 mDC) and 5 months (range 3–14) for the 11 DTH-negative patients (P < 0.001). The in vitro evaluation of gamma IFN-secreting T-cells in 10 patients showed good correlation with both DTH (75%) and clinical outcome (70%). Conclusion Vaccination using DC pulsed with ATL/ATH and KLH in advanced melanoma patients is well tolerated and can induce a clinical response, especially when mDC are used. Successful immunization, verified by positive DTH, leads to longer survival.

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 (P0.001), FasL expression was detected in 45% cells from melanomas (P0.001) 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.

Adoptive immunotherapy with tumor infiltrating lymphocytes (TIL) and interleukin (IL)-2 is reasonably effective in the treatment of patients with advanced melanoma. However, theoretically it should be of greater benefit as adjuvant therapy, especially in high-risk stages (resected stages III and IV). In a preliminary study, 25 patients (aged 23-72 years) with stage III-IV melanoma who underwent resection of metachronous metastases were reinfused with TIL cultivated and expanded in vitro with IL-2 from surgically removed metastases. IL-2 (starting dose 12 x 10 6 IU/m 2 ) was co-administered as a continuous infusion according to West's scheme. A total of 8/22 (36.3%) evaluable patients were disease-free (DF) at a median follow-up of 5 years. DF survival (DFS) and overall survival (OS) rates were 44% and 37%, respectively, at 2 years, and 52% and 45% at 3 years. The CNS was the only site of disease recurrence in 57% of patients who relapsed. DF patients received a higher median dose of IL-2 than those who progressed (total dose 110 x 10 6 versus 86 x 10 6 IU/m 2 , respectively). The progressive reduction in IL-2 dosage allowed all patients to complete treatment without permanent grade 4 toxicity. Analysis of tumor immunosuppression factors in lymphocytes inside the tumor (TCR ζ and e chains, p56 lck , FAS, and FAS-ligand) confirmed that the immunologic potential of TIL, depressed at the time of metastasectomy, was significantly restored after in vitro culture with IL-2. Adoptive immunotherapy with TIL and IL-2 could improve DFS and OS, although further work is required to determine its role in the treatment of patients with high-risk melanoma.

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.

Twenty-one patients with advanced, pretreated colorectal cancer in disease progression were entered in a phase II study to investigate the use of 5-fluorouracil (5FU) + leucovorin with subcutaneous Interleukin-2 + alpha interferon (alpha-IFN). Eighteen of these patients were evaluable for response to treatment: 1 partial response (PR) (duration 8 months), 9 stable disease (SD) (median duration of 6.5 months, range 2-15) and 8 progressive disease (PD). The PR patient survived for 15 months, the SD patients for a median of 11 months and 8 months for PD patients. Toxicity evaluated in the 21 patients reached grade 4 for mucositis in two cases. Grade 3 toxicity was observed more frequently for fever (52.3%) and diarrhea (33.3%) and was most probably the result of the combined side-effect of chemotherapy and the biological response modifiers (BRMs). Treatment was, for the most part, carried out on an out-patient basis as originally planned. In 15 patients tests were carried out to verify whether any immuno-activation had taken place. Significant increases were found during the course of therapy regarding cluster of differentiation activation (HLA-DR, CD71, CS25). Different curves were observed during the course of treatment with respect to the CD8 value, which proved higher in SD patients than in PD patients. Our study would seem to suggest that the addition of BRMs to 5FU + leucovorin could increase survival. The next step, however, must be to determine lower doses of IL-2 for subcutaneous administration in order to reduce toxicity but maintain the same immunostimulation.

8591 Background: One recently developed strategy in treating patients with stage IV melanoma consists of dendritic cell (DC) vaccination. This therapy has variable results, based on patients’ immun...

e13029 Background: Cytolytic T lymphocytes (CTL) able to kill tumor cells are efficiently induced by Dendritic Cells (DC); however, immunosuppressive factors, as CD4+CD25+FoxP3+ T lymphocytes (T-re...

TPS311 Background: Dendritic cells (DCs) play a central role in the induction of specific antigen recognition and tumor cell killing. Therapeutic vaccination with DCs is based on the potential for the in vitro cultivation and maturation of DC precursors from peripheral blood over a maximum of 7 days using GM-CSF and IL-4 in order to avoid the influence of immunosuppressive mechanisms present in tumor tissue. Among these, attention has recently focused on CD4+CD25+FoxP3+ T lymphocytes (T-regs). It has been seen that daily low-dose temozolomide (TMZ) induces lymphopenia, with a consequent reduction in T-regs (Su YB, J Clin Oncol, 2004). Furthermore, in a preliminary experience, we evaluated the expression of tumor endothelial marker 8 (TEM8) as a predictive marker of immunological and clinical response in mature DCs (mDCs) (Venanzi FM, Cancer Immunol Immunother, 2009). Following our experience of a vaccination trial using autologous mDCs pulsed with autologous tumor lysate (ATL) and keyhole limpet hemocyani...

9042 Background: Dendritic cells (DCs) play a crucial role in the interplay between innate and adaptive immune response towards cancer. The combination of immunotherapies with standard treatments for cancer could represent a further chance for advanced melanoma patients. In the literature, higher response rates than those normally obtained have been reported after second-line chemotherapy in patients with non small cell lung cancer pre-treated with vaccines and in patients with follicular B-cell lymphoma vaccinated with an anti-idiotype vaccine whilst in remission. On the basis of this data, we reviewed and updated the clinical results of our dendritic cell based vaccine clinical trial in stage IV melanoma patients. Methods: From December 2002 to 2007, 24 pre-treated metastatic melanoma patients were vaccinated with mature DCs (mDCs) pulsed with autologous tumor lysate (ATL) and keyhole limpet hemocyanin (KLH) followed by a 5-day treatment with low-dose subcutaneous Interleukin-2. Results: We observed 2 complete response (CR), 2 mixed response (MR), 5 partial response (PR), 4 stable disease (SD) and 11 progressive disease (PD) (overall response (OS) 37.5%; clinical benefit 54.1%). All 13 responders had delayed-type hypersensitivity (DTH) positivity to KLH, of whom 10 also showed positivity to the lysate. Eleven (45.8%) of the 24 patients underwent further lines of treatment (5 chemotherapy [CT], 3 surgery [S], 4 biotherapy, 2 radiotherapy [RT] and 4 biochemotherapy [BioCT]) after stopping vaccination (8 due to progression and 3, in SD, because all of their lysate had been used). Of these 11 patients, 2 obtained CR (1 RT, 1 S), 5 PR (3 BioCT, 2 S) for an OR of 63.6%, 1 SD (BioCT) and 3 showed PD as the best response to subsequent therapies, with a median OS of 30 months (range 16–52). Of the 3 SD patients who were forced to stop vaccine treatment, 1 had CR following RT and 2 progressed. Conclusions: Metastatic melanoma responds poorly to standard therapy, in particular after first-line treatment. Vaccination could enhance clinical response to subsequent third- or fourth-line therapies, thus prolonging overall survival. No significant financial relationships to disclose.

The feasibility of techniques developed for isolating and culturing human mammary epithelial cells of malignant origin was confirmed in 136 primary breast cancers, 116 hypodermal metastases, and 8 metastatic lymph nodes. In 115 (84%) primary breast cancers and in 81 (70%) hypodermal recurrences we observed a good in vitro cellular proliferation. These proliferating cells, at the second passage, were used for a clonal assay suitable for quantitating drug sensitivity. With this clonal assay median cloning efficiencies of 14% and 6% were obtained respectively in primaries and in skin recurrences. We examined the in vitro response to different drugs and confirmed the test's ability to detect heterogeneity in response to same drugs (doxorubicin, 4'-epidoxorubicin, vinblastine, cis platinum, and idarubicinol) among the different breast carcinoma cultures as well as heterogeneity among subpopulations within a single carcinoma.

20017 Background: Although dendritic cells (DC) play a fundamental role in immunoresponse, inside tumor tissue they are highly ineffective, probably because of tumor immunosuppression. Despite this...

7253 Background: Small cell lung cancer (SCLC) accounts for 20–25% of all lung cancers and often presents neuroendocrine characteristics. Neuron-specific enolase (NSE) is the most widely used marke...

Background Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissues, where they have the task of priming naive T-cells. In the present study in vivo DC migration was investigated within the context of a clinical trial of antitumor vaccination. In particular, we compared the migration activity of mature Dendritic Cells (mDC) with that of immature Dendritic Cells (iDC) and also assessed intradermal versus subcutaneous administration.

Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissue, where they prime naive T cells. In the present study DC were differentiated from PBMC obtained by leukapheresis (7-day culture of adherent cells with IL-4 and GM-CSF). On day 7 the DC were defined as immature DC (iDC). After a transient (3 h) and continuous (48 h) stimulation with a cocktail of cytokines (IL-1β, IL-6, TNF-α, PGE2), the iDC were defined as transientlyDC (tsDC) and mature DC (mDC), respectively. During a phase I-II clinical vaccination trial for patients with advanced melanoma and renal carcinoma, we evaluated the migration ability of iDC, tsDC and mDC, and compared intradermal (id) and subcutaneous (sc) administration. DC were labelled with 99Tc-HMPAO or 111In-Oxine and the presence of labelled DC was detected in regional lymph nodes up to a maximum of 72 h after inoculation.