Biomedical subjects

Paola Zanovello

Publications and source records attributed to Paola Zanovello.

At least 19 recordsLinked to original sources

PubMed2006

A gene expression signature associated with survival in metastatic melanoma.

BACKGROUND: Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival. METHODS: Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM) to identify genes associated with patient survival, and supervised principal components (SPC) to determine survival prediction. RESULTS: SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival. CONCLUSION: The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells.

Journal Article↗

PubMed2006

Survivin in esophageal cancer: An accurate prognostic marker for squamous cell carcinoma but not adenocarcinoma.

We quantified the expression of survivin, both as mRNA in real-time PCR and protein in immunohistochemistry, in tumor samples of 112 patients with esophageal cancer (56 squamous cell carcinomas and 56 adenocarcinomas). Overall survival of squamous cell carcinoma patients with high survivin mRNA levels was significantly less than that of patients with low survivin mRNA levels (p = 0.0033). Distribution pattern of survivin (nuclear vs. cytoplasmic or mixed) was not correlated to survival, while the extent of immunostaining was significantly correlated to survivin mRNA values (p = 0.016) and had prognostic relevance in univariate analysis (p = 0.0012). Cox's proportional-hazard regression model showed that tumor survivin expression in esophageal squamous cell carcinoma was the most important prognostic factor, independent of tumor stage and other histopathological factors, both as mRNA relative value (p = 0.0259) and protein immunostaining (p = 0.0147). In esophageal adenocarcinoma, survivin expression and pattern of distribution had no prognostic relevance. Thus, quantifying survivin expression provides a prognostic marker only for esophageal squamous tumors.

Adenocarcinoma↗

PubMed2006

Common cancer biomarkers.

There is an increasing interest in complementing conventional histopathologic evaluation with molecular tools that could increase the sensitivity and specificity of cancer staging for diagnostic and prognostic purposes. This study strove to identify cancer-specific markers for the molecular detection of a broad range of cancer types. We used 373 archival samples inclusive of normal tissues of various lineages and benign or malignant tumors (predominantly colon, melanoma, ovarian, and esophageal cancers). All samples were processed identically and cohybridized with an identical reference RNA source to a custom-made cDNA array platform. The database was split into training (n = 201) and comparable prediction (n = 172) sets. Leave-one-out cross-validation and gene pairing analysis identified putative cancer biomarkers overexpressed by malignant lesions independent of tissue of derivation. In particular, seven gene pairs were identified with high predictive power (87%) in segregating malignant from benign lesions. Receiver operator characteristic curves based on the same genes could segregate malignant from benign tissues with 94% accuracy. The relevance of this study rests on the identification of a restricted number of biomarkers ubiquitously expressed by cancers of distinct histology. This has not been done before. These biomarkers could be used broadly to increase the sensitivity and accuracy of cancer staging and early detection of locoregional or systemic recurrence. Their selective expression by cancerous compared with paired normal tissues suggests an association with the oncogenic process resulting in stable expression during disease progression when the presently used differentiation markers are unreliable.

Biomarkers, Tumor↗

PubMed2006

Predicting tumor outcome following cancer vaccination by monitoring quantitative and qualitative CD8+ T cell parameters.

Identification of reliable surrogate predictors for evaluation of cancer vaccine efficacy is a critical issue in immunotherapy. We analyzed quantitative and qualitative CD8+ T cell parameters in a large pool of BALB/c mice that were DNA-vaccinated against P1A self tumor-specific Ag. After immunization, mice were splenectomized and kept alive for a subsequent tumor challenge to correlate results of immune monitoring assays with tumor regression or progression in each individual animal, and to assess the prognostic value of the assays. The parameters tested were 1) percentage of in vivo vaccine-induced tumor-specific CD8+ T cells; 2) results of ELISPOT tests from fresh splenocytes; 3) percentage of tumor-specific CD8+ T cells in culture after in vitro restimulation; 4) in vitro increase of tumor-specific CD8+ T cell population expressed as fold of expansion; and 5) antitumor lytic activity of restimulated cultures. Except for the ELISPOT assay, each parameter tested was shown by univariate statistical analysis to correlate with tumor regression. However, multivariate analysis revealed that only in vitro percentage of Ag-specific CD8+ T cells was an independent prognostic factor that predicted tumor outcome. These findings should be considered in the design of new immune monitoring systems used in cancer immunotherapy studies.

Animals↗

PubMed2006

Leukocyte infiltration in cancer creates an unfavorable environment for antitumor immune responses: a novel target for therapeutic intervention.

The interaction between tumor cells and the nearby environment is being actively investigated to explore how this interplay affects the initiation and progression of cancer. Host-tumor relationship results in the production of pro-inflammatory cytokines and chemokines that promote the recruitment of leukocytes within and around developing neoplasms. Cancer cells, together with newly recruited tumor-infiltrating cells, can also activate fibroblast and vascular responses, thus resulting in a chronic microenvironment perturbation. In this complex scenario, interactions between innate and adaptive immune cells can be disturbed, leading to a failure of immune-mediated tumor recognition and destruction. On the basis of the recent awareness about tumor promotion and immune deregulation by immune/inflammatory cells, novel anti-cancer strategies can be exploited.

Animals↗

PubMed2006

Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.

Active suppression of tumor-specific T lymphocytes can limit the efficacy of immune surveillance and immunotherapy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+IL-4 receptor alpha+ (CD11b+IL-4Ralpha+), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-gamma released from T lymphocytes. CD11b+IL-4Ralpha+ cells produced IL-13 and IFN-gamma and integrated the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits were active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors had detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumor-induced immune dysfunctions.

Animals↗

PubMed2005

Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination.

Active suppression of tumor-specific T lymphocytes can limit the immune-mediated destruction of cancer cells. Of the various strategies used by tumors to counteract immune attacks, myeloid suppressors recruited by growing cancers are particularly efficient, often resulting in the induction of systemic T lymphocyte dysfunction. We have previously shown that the mechanism by which myeloid cells from tumor-bearing hosts block immune defense strategies involves two enzymes that metabolize L-arginine: arginase and nitric oxide (NO) synthase. NO-releasing aspirin is a classic aspirin molecule covalently linked to a NO donor group. NO aspirin does not possess direct antitumor activity. However, by interfering with the inhibitory enzymatic activities of myeloid cells, orally administered NO aspirin normalized the immune status of tumor-bearing hosts, increased the number and function of tumor-antigen-specific T lymphocytes, and enhanced the preventive and therapeutic effectiveness of the antitumor immunity elicited by cancer vaccination. Because cancer vaccines and NO aspirin are currently being investigated in independent phase I/II clinical trials, these findings offer a rationale to combine these treatments in subjects with advanced neoplastic diseases.

Animals↗

PubMed2005

Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes.

PURPOSE: Nemorubicin (3'-deamino-3'-[2''(S)-methoxy-4''-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682. EXPERIMENTAL DESIGN: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively. RESULTS: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts. CONCLUSIONS: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.

Animals↗

PubMed2005

Polarized monocyte response to cytokine stimulation.

BACKGROUND: Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate. RESULTS: Circulating CD14+ MPs were exposed to bacterial lipopolysaccharide (LPS) followed by exposure to an array of cytokines, chemokines and soluble factors involved in the immune response. Gene expression was studied by global transcript analysis. Two main classes of cytokines were identified that induced a classical or an alternative pathway of MP activation. Expression of genes affected by NFkappaB activation was most predictive of the two main classes, suggesting that this pathway is a fundamental target of cytokine regulation. As LPS itself induces a classical type of activation, the most dramatic modulation was observed toward the alternative pathway, suggesting that a broad array of cytokines may counteract the pro-inflammatory effects of bacterial components. CONCLUSIONS: This analysis is directly informative of the primary effect of individual cytokines on the early stages of LPS stimulation and, therefore, may be most informative of the way MP maturation may be polarized at the early stages of the immune response.

Adult↗

PubMed2005

Regulation of immune responses by L-arginine metabolism.

L-Arginine is an essential amino acid for birds and young mammals, and it is a conditionally essential amino acid for adult mammals, as it is important in situations in which requirements exceed production, such as pregnancy. Recent findings indicate that increased metabolism of L-arginine by myeloid cells can result in the impairment of lymphocyte responses to antigen during immune responses and tumour growth. Two enzymes that compete for L-arginine as a substrate - arginase and nitric-oxide synthase - are crucial components of this lymphocyte-suppression pathway, and the metabolic products of these enzymes are important moderators of T-cell function. This Review article focuses on the relevance of L-arginine metabolism by myeloid cells for immunity under physiological and pathological conditions.

Animals↗

PubMed2004

Therapeutic effectiveness of recombinant cancer vaccines is associated with a prevalent T-cell receptor alpha usage by melanoma-specific CD8+ T lymphocytes.

Definition of immune variables that correlate with the antitumor activity of cancer vaccines is critical for monitoring immunotherapy protocols. To define surrogate end points predictive of the therapeutic efficacy of recombinant vaccines based on melanoma antigen tyrosinase-related protein (TRP)-2, we evaluated several properties of antigen-specific CD8(+) T lymphocytes in single mice undergoing either prophylactic or therapeutic immunization. Predictive markers for the efficacy of genetic vaccination were identified in the prophylactic model used. Interestingly, the number of tetramer(+) CD8(+) T lymphocytes expanded in vitro after a single cycle of stimulation with the immunodominant TRP-2 peptide was of the highest predictive value. In the therapeutic model, no variable examined at a single mouse level predicted the long-term therapeutic effect. Mice that survived did not show the highest expansion of antigen-specific lymphocytes or the more functionally active effectors, ex vivo or after in vitro culture with the peptide antigen. Successful therapy correlated strictly with the skewing of the T-cell receptor repertoire of tetramer-sorted, TRP-2-specific CD8(+) T lymphocytes, which showed a preferential alpha chain usage with a common CDR3 region.

Adenoviridae↗

PubMed2004

Melanoma-restricted genes.

Human metastatic cutaneous melanoma has gained a well deserved reputation for its immune responsiveness. The reason(s) remain(s) unknown. We attempted previously to characterize several variables that may affect the relationship between tumor and host immune cells but, taken one at the time, none yielded a convincing explanation. With explorative purposes, high-throughput technology was applied here to portray transcriptional characteristics unique to metastatic cutaneous melanoma that may or may not be relevant to its immunogenic potential. Several functional signatures could be identified descriptive of immune or other biological functions. In addition, the transcriptional profile of metastatic melanoma was compared with that of primary renal cell cancers (RCC) identifying several genes co-coordinately expressed by the two tumor types. Since RCC is another immune responsive tumor, commonalities between RCC and melanoma may help untangle the enigma of their potential immune responsiveness. This purely descriptive study provides, therefore, a map for the investigation of metastatic melanoma in future clinical trials and at the same time may invite consideration of novel therapeutic targets.

Journal Article↗

PubMed2003

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase.

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.

Animals↗

PubMed2003

Individual analysis of mice vaccinated against a weakly immunogenic self tumor-specific antigen reveals a correlation between CD8 T cell response and antitumor efficacy.

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.

Adjuvants, Immunologic↗

PubMed2003

Derangement of immune responses by myeloid suppressor cells.

In tumor-bearing mice and cancer patients, tumor progression is often associated with altered hematopoiesis leading to the accumulation of myeloid cells. Extensive studies in preclinical models indicate that these cells share the CD11b and the Gr-1 markers, possess a mixed mature-immature myeloid phenotype, and are responsible for the induction of T-cell dysfunctions, both tumor-specific and nonspecific. Moreover, CD11b(+)Gr-1(+) myeloid cells are described under different unrelated situations associated with temporary impairment of the T-lymphocyte reactivity. This review examines recent findings on the nature, properties, and mechanisms of action of these myeloid suppressor cells (MSCs).

Animals↗

PubMed2003

The cytotoxic T-lymphocyte response against a poorly immunogenic mammary adenocarcinoma is focused on a single immunodominant class I epitope derived from the gp70 Env product of an endogenous retrovirus.

The TS/A mouse mammary adenocarcinoma is a poorly immunogenic tumor widely used in preclinical models of cancer immunotherapy. CTLs have often been indicated as important in TS/A tumor destruction, but their generation in this model has been rarely studied, nor have their precise target(s) been identified. We hypothesized that the gp70 Env product of an endogenous murine leukemia virus could be a target antigen for TS/A-specific CTLs and investigated this possibility in four different TS/A cell lines engineered with the genes that encode IFN-alpha, IFN-gamma, interleukin-4, and B7.1, respectively. All tumor cell lines expressed gp70, albeit at different levels, as demonstrated by reverse transcription-PCR analysis. Transfected tumor cells exhibited a delayed growth in vivo, and partial tumor regression. Spleen cells from mice that displayed tumor regression had high percentages of CD8(+) T cells that were specifically stained with L(d) tetramers loaded with gp70(423-431), the antigenic epitope of gp70 protein. Mixed leukocyte-peptide and mixed leukocyte-tumor cultures, set up by stimulating splenocytes with the immunogenic peptide and with transfected TS/A tumor cells, respectively, resulted in similar large increases in tetramer-reactive CD8(+) T cells and showed high lytic activity specific for gp70(423-431). Finally, in a Cold Target Inhibition assay, lytic activity of a mixed leukocyte-tumor culture was inhibited in an overlapping fashion by both the TS/A line used for restimulation and 293L(d) cells loaded with gp70(423-431) peptide, but not by 293L(d) cells pulsed with an irrelevant H-2 L(d) epitope, thus demonstrating that all or most of the cytotoxic activity was directed exclusively against this antigenic epitope.

Adenocarcinoma↗

PubMed2003

IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice.

We previously demonstrated that a specialized subset of immature myeloid cells migrate to lymphoid organs as a result of tumor growth or immune stress, where they suppress B and T cell responses to Ags. Although NO was required for suppression of mitogen activation of T cells by myeloid suppressor cells (MSC), it was not required for suppression of allogenic responses. In this study, we describe a novel mechanism used by MSC to block T cell proliferation and CTL generation in response to alloantigen, which is mediated by the enzyme arginase 1 (Arg1). We show that Arg1 increases superoxide production in myeloid cells through a pathway that likely utilizes the reductase domain of inducible NO synthase (iNOS), and that superoxide is required for Arg1-dependent suppression of T cell function. Arg1 is induced by IL-4 in freshly isolated MSC or cloned MSC lines, and is therefore up-regulated by activated Th2, but not Th1, cells. In contrast, iNOS is induced by IFN-gamma and Th1 cells. Because Arg1 and iNOS share L-arginine as a common substrate, our results indicate that L-arginine metabolism in myeloid cells is a potential target for selective intervention in reversing myeloid-induced dysfunction in tumor-bearing hosts.

Animals↗

PubMed2003

L-arginine metabolism in myeloid cells controls T-lymphocyte functions.

Although current attention has focused on regulatory T lymphocytes as suppressors of autoimmune responses, powerful immunosuppression is also mediated by a subset of myeloid cells that enter the lymphoid organs and peripheral tissues during times of immune stress. If these myeloid suppressor cells (MSCs) receive signals from activated T lymphocytes in the lymphoid organs, they block T-cell proliferation. MSCs use two enzymes involved in arginine metabolism to control T-cell responses: inducible nitric oxide synthase (NOS2), which generates nitric oxide (NO) and arginase 1 (Arg1), which depletes the milieu of arginine. Th1 cytokines induce NOS2, whereas Th2 cytokines upregulate Arg1. Induction of either enzyme alone results in a reversible block in T-cell proliferation. When both enzymes are induced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T lymphocytes to undergo apoptosis. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. This Opinion is the second in a series on the regulation of the immune system by metabolic pathways.

Animals↗