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Biomedical subjects

G Parmiani

Publications and source records attributed to G Parmiani.

At least 19 recordsLinked to original sources
PubMed2006

Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been and is still widely used as an adjuvant in clinical trials of vaccination with autologous tumor cells, peptides and/or dendritic cells in a variety of human neoplasms. This cytokine was administered either as product of gene-transduced tumor cells or as recombinant protein together with the vaccine given subcutaneously or intradermally. Results of these trials were heterogeneous in terms of induction of vaccine-specific immune response and of clinical response. Though in some of these studies GM-CSF appeared to help in generating an immune response, in others no effect or even a suppressive effect was reported. Here, we review the literature dealing with the immune adjuvant activity of GM-CSF both in animal models and clinical trials. As a consequence of such analysis, we conclude that GM-CSF may increase the vaccine-induced immune response when administered repeatedly at relatively low doses (range 40-80 microg for 1-5 days) whereas an opposite effect was often reported at dosages of 100-500 microg. The potential mechanisms of the GM-CSF-mediated immune suppression are discussed at the light of studies describing the activation and expansion of myeloid suppressor cells by endogenous tumor-derived or exogenous GM-CSF.

Adjuvants, Immunologic↗
PubMed2006

Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma.

Activating BRAF or NRAS mutations have been found in 80% of human sporadic melanomas, but only one of these genetic alterations could be detected in each tumour. This suggests that BRAF and NRAS 'double mutants' may not provide advantage for tumour growth, or may even be selected against during tumorigenesis. However, by applying mutant-allele-specific-amplification-PCR method to short-term melanoma lines, one out of 14 tumours was found to harbour both BRAFV600E and the activating NRASQ61R mutations. On the other hand, analysis of 21 melanoma clones isolated by growth in soft agar from this tumour indicated that 16/21 clones harboured a BRAFV600E, but were wild-type for NRAS, whereas the remaining had the opposite genotype (NRASQ61R/wild-type BRAF). When compared to BRAFV600E clones, NRASQ61R clones displayed reduced growth in soft agar, but higher proliferative ability in vitro in liquid medium and even in vivo after grafting into SCID/SCID mice. These data suggest that NRAS and BRAF activating mutations can coexist in the same melanoma, but are mutually exclusive at the single-cell level. Moreover, the presence of NRASQ61R or BRAFV600E is associated with distinct in vitro and in vivo growth properties of neoplastic cells.

Animals↗
PubMed2003

Vaccination of patients with solid tumours.

The molecular characterisation of human tumour antigens recognised by T cells has provided new impetus for immunisation of patients bearing tumours expressing well-defined antigens. After evaluating the immunogenicity of the new, molecularly characterised antigens in vitro, several clinical studies were conducted to assess the in vivo immunogenicity and the clinical efficacy of vaccines including these antigens. The findings generated by trials based on the administration of peptides or DNA-encoding antigens are discussed to highlight the limits of this therapeutic approach; however, this approach has resulted in some complete and durable regressions, although still in a unsatisfactory small number of cases (5-25%). The recent use of dendritic cells loaded ex vivo with tumour antigens suggests that a high frequency of tumour-specific immune responses can be achieved. Possible means of overcoming the clinical limits and improving the outcome of previous studies are also discussed.

Animals↗
PubMed2002

Identification of a novel gp100/pMel17 peptide presented by HLA-A*6801 and recognized on human melanoma by cytolytic T cell clones.

Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.

Amino Acid Sequence↗
PubMed2001

Phases of apoptosis of melanoma cells, but not of normal melanocytes, differently affect maturation of myeloid dendritic cells.

In this study, we investigated whether maturation of monocyte-derived myeloid dendritic cells (DCs) is differentially affected by the uptake of dying human melanoma cells in distinct phases of apoptosis. Maturation of monocyte-derived DCs, as documented by phenotype analysis and T-cell immunostimulatory activity, was inhibited by phagocytosis of dying melanoma cells containing a large fraction of cells in early apoptosis (Annexin-V+ and propidium iodide-) but promoted by the same tumors when in late apoptosis/secondary necrosis (Annexin-V+ and propidium iodide+) or when dying by primary necrosis. These opposite effects on DC maturation were observed after the uptake of early or late apoptotic cells from most vertical growth phase primary tumors and all metastases but not after the uptake of dying cells from a radial growth phase primary tumor or normal adult melanocytes. Inhibition of DC maturation by early apoptotic melanoma cells correlated with expression of interleukin-10 in neoplastic cells and was prevented by preincubating the tumor cells with a neutralizing antibody to interleukin-10 before tumor uptake by DCs. Cross-presentation of the melanoma-associated antigen gp100(209-217) to peptide-specific CTLs by HLA-A*0201+ DCs was achieved 48-72 h after phagocytosis of HLA-A*0201- melanoma cells in apoptosis, or primary necrosis, but only when tumor necrosis factor-alpha was added to DCs 4 h after the initiation of tumor phagocytosis. These results suggest that phases of apoptosis and neoplastic transformation affect maturation of myeloid DCs that take up dying cells of the melanocyte lineage. However, neoplastic cells in late apoptosis, or even in primary necrosis, induce only a partial DC differentiation not sufficient to achieve cross-presentation of tumor antigens to CTLs unless further DC maturation is promoted by additional signals. These results suggest a novel mechanism of tumor escape that may prevent the development of antitumor immunity through the maturation block induced in DCs by neoplastic cells in the early phase of apoptosis.

Adult↗
PubMed2001

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation.

Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.

Adult↗
PubMed2001

Interferon adjuvant to radical nephrectomy in Robson stages II and III renal cell carcinoma: a multicentric randomized study.

PURPOSE: Because interferon gave promising results in the management of metastatic renal cell carcinoma in the 1980s, a multicentric randomized controlled trial was planned to compare adjuvant recombinant interferon alfa-2b (rIFNalpha2b) with observation after radical nephrectomy in patients with Robson stages II and III renal cell carcinoma. Overall and event-free survival were to be evaluated together with prognostic factors. PATIENTS AND METHODS: Overall and event-free survival curves for 247 patients (124 controls and 123 treated) were estimated by the Kaplan-Meier method and compared using the log-rank test. Cox's multiple regression models were adopted to perform a joint analysis of treatment and prognostic factors. RESULTS: The 5-year overall and event-free survival probabilities were 0.665 and 0.671, respectively, for controls and 0.660 and 0.567, respectively, for the treated group; the differences were not statistically significant (2P = .861 for overall and 2P = .107 for event-free survival with the log-rank test). Regarding prognostic factors, only grade, pT, and pN demonstrated a significant prognostic role. First-order interactions of treatment with pT and pN category were investigated; a significant interaction was found between pN and treatment. A harmful effect of rIFNalpha2b in the 97 treated pN0 patients and a protective effect in the 13 treated pN2/pN3 patients were statistically significant. CONCLUSION: Adjuvant rIFNalpha2b is not indicated after radical nephrectomy for renal cell carcinoma. The protective effect in the small group of pN2/pN3 patients requires further investigation.

Adult↗
PubMed2001

Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells.

Members of the heat shock protein 70 (HSP70) family display a broad cellular localization and thus bind a repertoire of chaperoned peptides potentially derived from proteins of different cellular compartments. In this report, we show that HSP70 purified from human melanoma can activate T cells recognizing melanoma differentiation antigens in an antigen- and HLA class I-dependent fashion. HLA class I-restricted anti-melanoma T cells were susceptible to MHC-restricted, HSP70-dependent stimulation, indicating that HSP70 complexed peptides were able to gain access to the class I HLA presentation pathway. In addition, MHC matching between the melanoma cells used as a source of HSP and the responding T cells were not required, indicating that HSP70 activation may occur across MHC barriers. Besides the MHC-restricted and peptide-dependent activation pathway, HSP70 with no endogenous complexed peptides or HSP70 purified from antigen-negative cells was also able to induce IFN-gamma release by antimelanoma T cells by a MHC-independent mechanism. In this case, however, higher doses of HSP70 were required. The capacity to activate class I-restricted, antitumor T cells as well as antigen-presenting cells, together with the finding that the HSP70 chaperoned peptide repertoire includes melanoma-shared epitopes, holds promise for a HSP70-based cancer vaccine.

Antigen-Presenting Cells↗
PubMed2001

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes.

In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-gamma release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients.

B7-1 Antigen↗
PubMed2001

Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy.

The effect of local and systemic delivery of the angiostatin gene on human melanoma growth was studied in nude mice. Liposome-coated plasmids carrying the cDNA coding for murine and human angiostatin (CMVang and BSHang) were injected weekly, locally or systemically, in mice transplanted with melanoma cells. The treatment reduced melanoma growth by 50% to 90% compared to that occurring in control animals treated with liposome-coated plasmid carrying the lacZ gene or in untreated controls. The growth of both locally injected and controlateral uninjected tumors in mice bearing two melanoma grafts was significantly suppressed after intratumoral treatment. Tumor growth inhibition was also observed in mice treated by intraperitoneal delivery, suggesting that angiostatin gene therapy acts through a systemic effect. Both melanoma growth suppression and delay in the onset of tumor growth were observed in treated mice. PCR performed on tumors and normal tissues showed that the lipofected DNA was present in tissues from treated mice, and angiostatin expression was demonstrated by RT-PCR. Histopathological analysis of melanoma nodules revealed an increase in apoptotic cells and a reduction in vessel density in tumors from treated mice. Our results suggest that systemic, liposome-mediated administration of genes coding for antiangiogenic factors represents a promising strategy for melanoma treatment in humans.

Angiostatins↗
PubMed2001

Melanoma antigens and their recognition by T cells.

During the last 10 years many melanoma antigens recognized by T cells have been molecularly characterized. This review summarizes the main features of these antigens, including both classes I and II HLA-restricted peptides, and describes their classification into diverse groups according to the tissue distribution of the antigens. The different in vitro and in vivo immunogenicity of such antigens is then discussed leading to the conclusion that Melan-A/MART-1 is the strongest among those tested being frequently recognized by patients' T cells both in vitro and in vivo. However, no correlation was found between T-cell response of melanoma patients to Melan-A/MART-1 and clinical response when it was used for vaccination. Data are also presented that suggest, through an ex vivo analysis carried out with tetramers staining of melanoma-specific T cells, that only in a limited number of advanced patients does a specific immune response develop. This response, however, appears unable to effectively counteract metastatic melanoma growth.

Antigens, Neoplasm↗
PubMed2000

T cell responses in colorectal cancer patients: evidence for class II HLA-restricted recognition of shared tumor-associated antigens.

Few cases of anti-colon cancer specific T lymphocytes have been described so far. Moreover, the majority of these effectors were generated in vitro by stimulating PBMC from patients or healthy donors with peptides that were derived from proteins expressed and/or secreted by colon cancer tissue such as CEA, Mucin or Her-2/neu. The aim of our study was to evaluate the immunogenicity of colorectal carcinomas in an autologous setting. We exploited the antigen processing and presentation capacity of dendritic cells (DC) to establish an in vitro autologous system that can bypass the need of obtaining cultured tumor cells. DC were generated from the adherent monocyte fraction of PBMC taken from stage II/III colorectal cancer patients. A single cell suspension was prepared by mechanical and enzymatic disruption of the surgical specimens immediately after resection. DC were loaded with autologous tumor lysate, obtained by repeated freezing and thawing, before being used as stimulators for autologous PBL. HLA-class II restricted T cells that recognize the autologous tumor could be generated in a proportion of patients. The fine specificity of the anti-tumor T cells indicates that differentiation as well as tumor restricted antigens are expressed in colon cancer and that these antigens can evoke a class II HLA-restricted response in an autologous setting. Altogether these findings may open a new perspective for a DC based vaccination of colon cancer patients.

Antigens, Neoplasm↗
PubMed2000

Cytokines in cancer therapy.

Cytokines are crucial factors in the activation and development of immune response, including responses against tumor cells. Interleukin (IL)-2, a T-cell growth factor, has been largely used to activate T and NK cells in vivo and to maintain such an activation for therapeutic purposes. When given to patients, IL-2 was shown to cause clinical responses, especially in metastatic melanoma and renal cancer patients, though its mechanism of action could not be completely elucidated. Cytokines (IL-2, IL-12, GM-CSF) are also used as natural adjuvants of vaccines of various formulation to help in activating and maintaining an antitumor immune response. This review summarizes findings deriving from the use of cytokines in cancer therapy and provides insights into future approaches when a more appropriate use of cytokines, together with new vaccines, is likely to improve clinical outcome.

Adjuvants, Immunologic↗
PubMed2000

Murine granulocytes control human tumor growth in SCID mice.

Severe combined immunodeficient (SCID) mice generally do not reject allogeneic or xenogeneic organ grafts and represent a unique model for investigating in vivo the behaviour of both normal and neoplastic human cells. However, cells from human primary tumors often do not grow in SCID mice. We have previously shown that the major reaction of SCID mice to the engraftment of human peripheral blood leukocytes is a massive granulocyte recruitment into the site of transplantation. In this study, we have investigated the role of murine granulocytes in the control of human tumor cell growth in SCID mice. We report here that murine granulocytes infiltrate and delimit the human tumor mass and that treatment of SCID mice with anti-murine granulocyte antibody markedly improves the growth of human tumor cell lines of different origin through suppression of the host granulocyte reaction. This finding provides a new tool for improving the human tumor take in SCID mice, thus opening new perspectives for a practical in vivo preclinical test of anti-tumor strategies. Moreover, this study, even with the limits of the known natural reaction against xenotransplants, further supports the importance of granulocytes in the control of tumor take and growth.

Animals↗
PubMed2000

Immunological gene therapy with ex vivo gene-modified tumor cells: a critique and a reappraisal.

Studies using animal models have demonstrated that transduction of genes encoding different cytokines into tumor cells results in a local recruitment of inflammatory cells that in turn can inhibit tumor growth. This is often accompanied by tumor antigen priming of the host immune system, which becomes resistant to subsequent challenge by the parental, untransduced tumor. Gene-transduced tumor cells have therefore been widely used as vaccines, although in the therapeutic setting their antitumor efficacy was limited to a few animal models. On the basis of this rationale, clinical studies were initiated, results of which are evaluated in this review to identify the reasons for their limited efficacy. We point out problems generated by the use of autologous versus allogeneic gene-transduced vaccines, by the choice of the appropriate cytokine(s), and by patient selection. Results of these studies are also compared with those obtained by peptide-based vaccines in similar groups of patients. Altogether, we conclude that improvements can be made in the construction of gene-modified vaccines by (1) using tumor cells known to express molecularly defined antigens, (2) introducing, in addition to genes encoding cytokines, genes encoding T cell costimulatory molecules, (3) increasing the amount of cytokine released locally by irradiated cells, and (4) coadministering adjuvant cytokines (IL-2 and IL-12) systemically in order to expand the T cell pool activated by vaccines.

Adjuvants, Immunologic↗
PubMed2000

T-cell recognition of melanoma-associated antigens.

In this review, we summarize the significant progress that has been made in the identification of melanoma-associated antigens (MAA) recognized by cytotoxic T-lymphocytes (CTL). These antigens belong to three main groups: tumor-associated testis-specific antigens (e.g. , MAGE, BAGE, and GAGE); melanocyte differentiation antigens (e.g., tyrosinase, Melan-A/MART-1); and mutated or aberrantly expressed molecules (e.g, CDK4, MUM-1, beta-catenin). Although strong CTL activity may be induced ex vivo against most of these antigens, often in the presence of excess cytokines and antigen, a clear understanding of the functional status of CTL in vivo and their impact on tumor growth, is still lacking. Several mechanisms are described that potentially contribute to tumor cell evasion of the immune response, suggesting that any antitumor efficacy achieved by immune effectors may be offset by factors that result ultimately in tumor progression. Nevertheless, most of these MAA are currently being investigated as immunizing agents in clinical studies, the conflicting results of which are reviewed. Indeed, the therapeutic potential of MAA has still to be fully exploited and new strategies have to be found in order to achieve an effective and long-lasting in vivo immune control of melanoma growth and progression.

Antigens, Neoplasm↗