The use of microarray technologies in clinical oncology.
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Journal Article↗
Biomedical subjects
F M Marincola
Publications and source records attributed to F M Marincola.
Degree of CD14 expression in melanoma infiltrating mononuclear phagocytes.
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Humans↗
IL-10 stimulatory effects on human NK cells explored by gene profile analysis.
The molecular mechanisms underlying the increase of natural killer (NK) cell anticancer activity mediated by interleukin (IL)-10 have not been elucidated. The aim of this study was to identify potential molecular mediators of IL-10 stimulatory effects by exploring the NK cell gene display induced by this cytokine. Gene profile was determined by high-throughput cDNA microarray and quantitative real-time PCR. In vitro, NK cells resting or conditioned with IL-10 were tested for cytotoxicity, migration and proliferation. IL-10 enhanced mRNA levels of cell activation/cytotoxicity-related genes (eg secretogranin, TIA-1, HMG-1, interferon-inducible genes) not upregulated by IL-2. In line with these findings, IL-10 increased NK cell in vitro cytotoxicity against Daudi cells. Unlike IL-2, IL-10 did not show any significant effect on NK cell in vitro proliferation and migration. However, gene profile analysis showed that IL-10 increased the expression of cell migration-related genes (eg L-selectin, vascular endothelium growth factor receptor-1, plasminogen activator, tissue; formyl peptide receptor, lipoxin A4 receptor), which might support a stimulatory effect not evident with the in vitro functional assay. Overall, gene profiling allowed us to formulate new hypotheses regarding the molecular pathways underlying the stimulatory effects of IL-10 on NK cells, supporting further investigation aimed at defining its role in cancer immune rejection.
Cell Movement↗
Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities.
Although there is increasing evidence that virus-specific cytotoxic-T-lymphocyte (CTL) responses play an important role in the control of human immunodeficiency virus (HIV) replication in vivo, only scarce CTL data are available for the ethnic populations currently most affected by the epidemic. In this study, we examined the CD8(+)-T-cell responses in African-American, Caucasian, Hispanic, and Caribbean populations in which clade B virus dominates and analyzed the potential factors influencing immune recognition. Total HIV-specific CD8(+)-T-cell responses were determined by enzyme-linked immunospot assays in 150 HIV-infected individuals by using a clade B consensus sequence peptide set spanning all HIV proteins. A total of 88% of the 410 tested peptides were recognized, and Nef- and Gag-specific responses dominated the total response for each ethnicity in terms of both breadth and magnitude. Three dominantly targeted regions within these proteins that were recognized by >90% of individuals in each ethnicity were identified. Overall, the total breadth and magnitude of CD8(+)-T-cell responses correlated with individuals' CD4 counts but not with viral loads. The frequency of recognition for each peptide was highly correlated with the relative conservation of the peptide sequence, the presence of predicted immunoproteasomal cleavage sites within the C-terminal half of the peptide, and a reduced frequency of amino acids that impair binding of optimal epitopes to the restricting class I molecules. The present study thus identifies factors that contribute to the immunogenicity of these highly targeted and relatively conserved sequences in HIV that may represent promising vaccine candidates for ethnically heterogeneous populations.
AIDS Vaccines↗
Biased epitope selection by recombinant vaccinia-virus (rVV)-infected mature or immature dendritic cells.
Recombinant expression vectors represent a powerful way to deliver whole antigens (Ags) for immunization. Sustained Ag expression in vector-infected dendritic cells (DC) combines Ag-specific stimulation with powerful costimulation and, simultaneously, through 'self-selection' of ad hoc epitopes broadens the scope of immunization beyond restrictions posed by individual patients' human leukocyte antigen (HLA) phenotype. In this study, therefore, we evaluated the efficiency of a recombinant vaccinia virus encoding the gp100/PMel17 melanoma Ag (rVV-gp100) to infect immature (iDC) or mature dendritic cells (mDC) derived from circulating mononuclear cells and the effect of infection on their status of maturation. In addition, we tested the ability of rVV-gp100-infected iDC and mDC to present the HLA-A*0201-associated gp100:209-217 epitope (g209). Irrespective of status of maturation, rVV-gp100 infection induced gp100 expression while only partially reversing the expression of some maturation markers. However, endogenous presentation of the wild-type g209 epitope was inefficient. The low efficiency was epitope-specific since infection of DC with rVV encoding a gp100 construct containing the modified gp100:209-217 (210M) (g209-2M) epitope characterized by high binding affinity for HLA-A*0201 restored efficient Ag presentation. Presentation of an HLA-class II-associated epitope and cytokine release by DC was not altered by rVV infection. Thus, Ag expression driven by rVV may be an efficient strategy for whole Ag delivery. However, since the effectiveness of Ag processing and presentation is subject to stringent HLA/epitope pairing, and for other yet undefined rules, the assumption that whole Ag delivery may circumvent HLA restriction is incorrect and recombinant expression vectors encoding well-characterized polyepitopic constructs may prove more effective.
Antigen Presentation↗
Use of laser scanning cytometry to study tumor microenvironment.
The study of phenomena occurring in the tumor microenvironment is a challenging task because of technical difficulties, particularly when dealing with hypocellular specimens. Laser scanning cytometry (LSC) is a new laboratory technology that has been recently introduced to overcome the limitations of other traditional technologies. By combining the properties and the advantages of flow cytometry (FC) and immunohistochemistry (IHC), LSC allows the investigator to obtain objective information on DNA content, protein expression and cellular localization is combination with morphological features. It has been already shown that LSC results are reliable compared to more traditional technologies, and its implementation in the clinical routine is under way. Its use in oncology, which is rapidly expanding, spans from apoptosis analysis to DNA content quantitation and tumor cell phenotyping. Here we describe the technology underlying this novel fluorescence-based device, review its use in oncology by dissecting the phenomena occurring in the tumor microenvironment and propose its application for the immunological follow-up of malignant lesions undergoing immunotherapeutic manipulation.
Animals↗
Immunotherapy against antigenic tumors: a game with a lot of players.
Our understanding of how immune responses are generated and regulated drives the design of possible immunotherapies for cancer patients. For that reason, we first describe briefly the actual immunological theories and their common perspectives about cancer vaccine development. Second, we describe cancer vaccines that are able to induce tumor-specific immune responses in cancer patients. However, these responses are not always followed by tumor rejection. At the end of the review, we discuss two possible reasons that might explain this dichotomy of cancer immunology. First, the immune response generated, although detectable, may not be quantitatively sufficient to reject the tumor. Second, the tumor microenvironment may modulate tumor cell susceptibility to the systemic immune response induced by the immunization. Finally, we discuss what, in our opinion, might be the best way to improve cancer vaccine strategies and how the relationship between the tumor and its surroundings might be studied in more details.
Antigens, Neoplasm↗
Advantages of mRNA amplification for microarray analysis.
Expanding applications of cDNA microarrays such as fine needle aspiration biopsy and laser capture microdissection necessitate the ability to perform arrays with minute starting amounts of RNA. While methods for amplifying RNA have been advocated, the fidelity of array results using amplified material has not been fully validated. Here we demonstrate preserved fidelity in arrays using one or two rounds of mRNA amplification, validated by downstream real-time quantitative PCR. In addition, the quality of the array data was superior to that obtained using total RNA. Based on these results, we recommend routine mRNA amplification for all cDNA microarray-based analysis of gene expression.
Animals↗
Melanoma antigen expression in serial fine-needle aspiration samples in patients with metastatic malignant melanoma participating in immunotherapy clinical trials: a preliminary look.
BACKGROUND: MART-1 and gp100 currently are utilized as targets in immunotherapy protocols for metastatic malignant melanoma (MMM). Enrollment of patients into ongoing peptide vaccination trials at the National Cancer Institute includes immunophenotyping of samples of metastatic lesions obtained by fine-needle aspiration (FNA). As therapy progresses, immunocytochemistry is performed on serial FNAs of metastatic lesions to monitor changes in antigen expression during treatment. It is theorized that antigen expression of melanoma cells may be diminished because of selective immunodestruction of tumor cells, or perhaps intentionally, to escape immunosurveillance. METHODS: Thirty-eight lesions from 33 patients were serially monitored for the expression of gp100 (clone HMB-45) and MART-1 (clone M2-7C10), using an avidin-biotin peroxidase technique. The staining intensity of tumor cells was scored on a scale of 0 to 3+, with the proportion of positive cells categorized as less than 25%, 25-50%, 50-75%, and greater than 75%. All lesions were examined within approximately 2 months after the start of peptide vaccination, providing a consistent timepoint for analysis. RESULTS: Using the Wilcoxon signed rank test, the authors found that there were no significant changes from baseline compared with 2 months later for quantitative antigen expression of HMB-45 or MART-1. However, there was a trend toward a decline in staining intensity of tumor cells for HMB-45. CONCLUSIONS: Preliminary results evaluating antigen expression during selective immunotherapy indicate a trend in the decline of staining intensity of tumor cells to HMB-45. Thus, although other studies have shown that peptide-based immunotherapy results in immune selection, this does not hinder the diagnostic utility of antibodies to HMB-45 and MART-1 in FNA samples of MMM.
Antigens, Neoplasm↗
Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes.
CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.
Antigen Presentation↗
Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.
Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.
Amino Acid Sequence↗
Short-term kinetics of tumor antigen expression in response to vaccination.
The melanoma patient's immune response to tumor has been extensively studied. Yet, the frequently observed coexistence of tumor-associated Ag (TAA)-specific T cells with their target cells in vivo remains unexplained. Loss of TAA expression might contribute to this paradox. We studied TAA expression in metastases by obtaining fine-needle aspirations from 52 tumor lesions in 30 patients with melanoma before and soon after immunotherapy. Limitations due to low amounts of starting material were overcome with a high fidelity antisense RNA amplification method. TAA expression was measured by quantitative real-time PCR of anti-sense RNA. Decrease in gp100/Pmel-17 TAA preceded tumor disappearance in several instances and could be best explained by immune selection because most patients had received gp100/Pmel-17-specific vaccination. Conversely, immune selection was absent in nonregressing lesions. These observations suggest that vaccination, when successful, triggers a broad inflammatory reaction that can lead to tumor destruction despite immune selection. Additionally, lack of clinical response might be attributed to lack of this initiating event rather than immune escape. This study provides an insight into the natural history of tumors and defines a strategy for the characterization of gene expression in tumors during therapy.
Adult↗
Kinetics of cytokine expression in melanoma metastases classifies immune responsiveness.
Production of cytokines (CKs) in the tumor micro-environment may modulate tumor-host interactions. However, pre-clinical models often provide conflicting data and there is no established role for CKs as modulators of the natural or treatment-related behavior of tumors. Serial sampling by fine-needle aspirates (FNAs) of identical metastases from patients affected with metastatic melanoma and undergoing IL-2-based vaccination allowed prospective measurement of IL-10, TGF-beta1, TGF-beta2 and IFN-gamma transcriptional levels assessed by quantitative real-time PCR. Thus, it was possible to prospectively document the expression of markers relevant to a given treatment and follow at the same time the clinical outcome of the lesions left in place. Eight of 27 metastatic lesions completely regressed in response to the treatment and 1 demonstrated >50% shrinkage. These regressions occurred after the follow-up FNA had been obtained. IL-10 transcript was differentially expressed in pre-treatment FNA of responding lesions (t-test p(2) = 0.002). During treatment, INF-gamma transcript levels significantly increased in regressing compared to non-regressing lesions (t-test p(2) = 0.03). These data suggest that the pre-treatment CK profile of the tumor micro-environment may determine clinical responsiveness to immune therapy. Furthermore, temporal changes in CK expression during treatment might describe the biological characteristics of an effective immune response.
Cytokines↗
Kinetics of TCR use in response to repeated epitope-specific immunization.
Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209-217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient's kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-gamma ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.
Cancer Vaccines↗
Unmasking cryptic epitopes after loss of immunodominant tumor antigen expression through epitope spreading.
The basis of intra-tumoral and systemic T cell reactivity toward cancer remains unclear. In particular the role that peripheral stimuli, whether endogenous or exogenous, play in shaping acquired immune response toward cancer remains poorly understood. In this study we document the surfacing of systemic immune reactivity toward a cryptic epitope from the MAGE-12 gene (MAGE-12:170-178), after temporary regression of a single melanoma metastasis, in response to gp100/PMel17-specific vaccination. This emergence was unlikely related to unusually high expression of MAGE-12 by the tumor, by the influence of analog epitopes to MAGE-12:170-178. Because MAGE-12 was unlikely to be expressed at sites other than the tumor, the demonstration of MAGE-12:170-178 reactivity in post- but not pre-vaccination circulating lymphocytes suggests that the systemically observed immune response was influenced by events induced by the vaccine at tumor site or draining lymph nodal areas. Possibly, as suggested by pre-clinical models, immunologic ignorance is the default response toward cancer in humans unless unusual stimulatory conditions occur in peripheral tissues. Surfacing of MAGE-12 specificity occurred in association with loss of gp100/PMel 17 targeted by the vaccine. This finding suggests that vaccinations might have effects beyond their intrinsic specificity and may trigger broader immune responses through epitope spreading by inducing changes within the tumor microenvironment. This may have important practical implication for the development of immunization strategies. Published 2001 Wiley-Liss, Inc.
Alleles↗
Discordant cellular and humoral immune responses to cytomegalovirus infection in healthy blood donors: existence of a Th1-type dominant response.
Previous studies have documented discordant cellular and humoral immune responses to subjects exposed to HIV-1, and that the nature of such responses may determine susceptibility and resistance to disease. We determined whether there is a spectrum of cellular versus humoral immunodominant responses to cytomegalovirus (CMV) infection. Blood samples from 50 healthy blood donors were tested for anti-CMV IgG antibodies and for proliferative responses of peripheral blood mononuclear cells (PBMC) to CMV antigens. Four patterns of immune responses to CMV were found: no detectable response (30%, Ab(-)/Tc(-)), anti-CMV IgG only (28%, Ab(+)/Tc(-)), both anti-CMV IgG and T lymphocyte proliferation to CMV antigens (18%, Ab(+)/Tc(+)), and, interestingly, T lymphocyte proliferation to CMV only (24%, Ab(-)/Tc(+)). To determine whether these immunodominant phenotypes correlate with the ability of PBMC to secrete IL-2 and IFN-gamma in response to CMV antigens, we found that a greater percentage of individuals with a T cell proliferative response to CMV antigens (Ab(-)/Tc(+) and Ab(+)/Tc(+)) responded with increased IL-2 (P = 0.001) and IFN-gamma levels (P = 0.002), compared to those without a proliferative response (Ab(-)/Tc(-) and Ab(+)/Tc(-)). Our data therefore demonstrate that different individuals exhibit different immunodominant patterns of response to CMV. In particular, some individuals who are exposed to CMV fail to develop an antibody response but do develop cellular immunity. Whether these different patterns predict susceptibility or resistance to CMV-induced disease remains to be determined.
Adult↗
T-cell-directed cancer vaccines: the melanoma model.
Significant advances in the understanding of the molecular basis for tumour/host interactions in humans have occurred in the last decade through studying patients with metastatic melanoma. This disease is characterised by its tendency to be modulated by immunologic factors. Furthermore, immunologic manipulation of the host with various systemic agents, in particular IL-2, frequently affects this natural phenomenon and can lead to complete rejection of cancer. By studying the cellular immunology occurring in patients undergoing immunotherapy, several tumour antigens (TA) and their epitopes recognised by human leukocyte antigen (HLA) class I-restricted cytotoxic T-lymphocytes (CTL) have been identified. Most of these TA are non-mutated molecules expressed by the majority of melanoma in vivo and most melanoma cell lines. In addition, unique minimal epitopic sequences play an immunodominant role in the context of specific HLA class I alleles. Since melanoma lesions from different patients often share expression of the same TA, and a minimal peptide sequence from a TA can cause immunologic changes in multiple patients, interest has grown in the development of TA-specific vaccines suitable for broad patient populations. Repeated in vitro stimulation of peripheral blood mononuclear cells (PBMC) with TA-derived epitopes can induce a high frequency of TA-reactive T-cells in melanoma patients. The same epitopes can also enhance TA-specific T-cell reactivity in vivo when administered subcutaneously in combination with Incomplete Freund's Adjuvant (IFA). Epitope-based vaccinations, however, have not shown strong clinical efficacy unless combined with IL-2 administration. Attempts to increase the efficacy of these vaccines have combined specialised antigen-presenting cells or the administration of whole TA through DNA- or RNA-based vaccines with the intention of increasing antigen presentation and processing. Save for scattered reports, however, the success of these approaches has been limited and T-cell-directed vaccination against cancer remains at a paradoxical standstill whereby anticancer immunisation can be induced but it is not sufficient, in most cases, to induce tumour regression. Using melanoma as the standard model for immunotherapy, we will review various methods of T-cell-directed vaccination, the monitoring and analysis of the resulting immune response, and several clinical trials in which cancer vaccines have successfully induced immunisation.
Antigens, Neoplasm↗
T-cell-directed cancer vaccines: mechanisms of immune escape and immune tolerance.
Recent clinical trials using vaccines directed toward tumour-associated antigens (TA) have shown the increasing capacity of vaccines to cause immunologic responses. In fact, strongly reactive TA-specific cytolytic T-lymphocytes and tumour-infiltrating lymphocytes (TIL) can be identified and expanded ex vivo from patients with metastatic melanoma vaccinated with melanoma-associated antigens. Paradoxically, this strong immunological response does not correlate with clinical tumour regression. Proposed mechanisms responsible for this glaring inconsistency are numerous and varied; systemic immunosuppressive as well as local mechanistic factors are implicated. In this review we will critically evaluate the possible mechanisms that allow tumours to escape immune destruction and be tolerated by the immune system. In addition, strategies that may allow further insight into the biology of tumour rejection are discussed, in the hope of deepening the understanding of this phenomenon and enhancing its therapeutic potential.
Cancer Vaccines↗