Valeria Giandomenico

Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells

T cells can be engineered to target tumor cells by transduction of tumor-specific chimeric receptors, consisting of an extracellular antigen-binding domain and an intracellular signaling domain. However, the peripheral blood of cancer patients frequently contains an increased number of T regulatory cells, which appear to inhibit immune reactivity. We have investigated the effects of T regulatory cells on chimeric T cells specific for the B-cell antigen CD19, as B-cell malignancies are attractive targets for chimeric T-cell therapy. When a CD19 single-chain Fv antibody was coupled to the CD3 zeta (ζ) chain, there was sharply reduced activity on exposure to T regulatory cells, measured by CD19+ target-induced proliferation and cytotoxicity. By contrast, expression in T cells of a chimeric receptor consisting of the intracellular portion of the CD28 molecule fused to the ζ-chain and CD19 single-chain Fv not only produced a higher proliferative response and an increased nuclear factor κB activation but also sustained these activities in the presence of T regulatory cells. These effects are seen whether the chimeric T cells are derived from normal donors or from patients with B-cell chronic lymphocytic leukemia, indicating the potential for clinical application in B cell malignancies.

Novel markers for enterochromaffin cells and gastrointestinal neuroendocrine carcinomas

The gene expression profile of metastasizing serotonin-producing neuroendocrine carcinomas, which arise from enterochromaffin cells in the jejunum and ileum, is still largely unknown. The aim of this study was to identify genes and proteins, which are preferentially expressed by neuroendocrine carcinoma and enterochromaffin cells and therefore potential novel biomarkers and/or therapeutic targets. Six carcinoma specimens and six normal ileal mucosas were profiled by Affymetrix microarrays. Advanced bioinformatics identified differentially and specifically expressed genes, which were validated by quantitative real-time-PCR on tumor cells extracted by laser capture microdissection and normal enterochromaffin cells extracted by immunolaser capture microdissection. We identified six novel marker genes for neuroendocrine carcinoma cells: paraneoplastic antigen Ma2 (PNMA2), testican-1 precursor (SPOCK1), serpin A10 (SERPINA10), glutamate receptor ionotropic AMPA 2 (GRIA2), G protein-coupled receptor 112 (GPR112) and olfactory receptor family 51 subfamily E member 1 (OR51E1). GRIA2 is specifically expressed by neuroendocrine carcinoma cells whereas the others are also expressed by normal enterochromaffin cells. GPR112 and OR51E1 encode proteins associated with the plasma membrane and may therefore become targets for antibody-based diagnosis and therapy. Hierarchical clustering shows high similarity between primary lesions and liver metastases. However, chemokine C-X-C motif ligand 14 (CXCL14) and NK2 transcription factor related locus 3 Drosophila (NKX2-3) are expressed to a lower level in liver metastases than in primary tumors and normal enterochromaffin cells, which implies a role in neuroendocrine carcinoma differentiation. In conclusion, this study provides a list of genes, which possess relatively specific expression to enterochromaffin and neuroendocrine carcinoma cells and genes with differential expression between primary tumors and metastases. We verified six novel marker genes that may be developed as biomarkers and/or therapeutic targets.

Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors

Well-differentiated small intestinal neuroendocrine tumors are rare malignancies. They arise from enterochromaffin cells and very little is known about differential microRNA (miRNA) expression. The aim of this study was to identify the miRNA profile of well-differentiated small intestinal neuroendocrine tumors, which may have a critical role in tumor development, progression and potentially develop miRNAs as novel clinical biomarkers. Specimens from two test groups, 24 small intestinal neuroendocrine tumor specimens at different stages of malignancy, are included in this study. Total RNA from the first test group, five primary tumors, five mesentery metastases and five liver metastases was hybridized onto the Affymetrix Genechip miRNA arrays to perform a genome-wide profile. The results were validated by using quantitative real-time PCR (QRT-PCR) and northern blot analyses. We then expanded the investigation to laser capture microdissected small intestinal neuroendocrine tumor cells and immuno-laser capture microdissected normal enterochromaffin cells of the first test group. Furthermore, a second test group, three primary tumors, three mesentery metastases and three liver metastases, was included in the study. Thus, two independent test groups validated the data by QRT-PCR. Moreover, we characterized nine miRNAs, five (miR-96, -182, -183, -196a and -200a), which are upregulated during tumor progression, whereas four (miR-31, -129-5p, -133a and -215) are downregulated. Several online software programs were used to predict potential miRNA target genes to map a number of putative target genes for the aberrantly regulated miRNAs, through an advanced and novel bioinformatics analysis. Our findings provide information about pivotal miRNAs, which may lead to further insights into tumorigenesis, progression mechanisms and novel therapeutic targets recognition.

Double-Detargeted Oncolytic Adenovirus Shows Replication Arrest in Liver Cells and Retains Neuroendocrine Cell Killing Ability

Background We have previously developed an oncolytic serotype 5 adenovirus (Ad5) with chromogranin-A (CgA) promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control. Methodology/Principal Findings Ad[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter. Conclusions/Significance A combination of transcriptional (promoter) and post-transcriptional (miRNA target) regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.

Paraneoplastic antigen Ma2 autoantibodies as specific blood biomarkers for detection of early recurrence of small intestine neuroendocrine tumors.

Background Small intestine neuroendocrine tumors (SI-NETs) belong to a rare group of cancers. Most patients have developed metastatic disease at the time of diagnosis, for which there is currently no cure. The delay in diagnosis is a major issue in the clinical management of the patients and new markers are urgently needed. We have previously identified paraneoplastic antigen Ma2 (PNMA2) as a novel SI-NET tissue biomarker. Therefore, we evaluated whether Ma2 autoantibodies detection in the blood stream is useful for the clinical diagnosis and recurrence of SI-NETs. Methodology/Principal Findings A novel indirect ELISA was set up to detect Ma2 autoantibodies in blood samples of patients with SI-NET at different stages of disease. The analysis was extended to include typical and atypical lung carcinoids (TLC and ALC), to evaluate whether Ma2 autoantibodies in the blood stream become a general biomarker for NETs. In total, 124 blood samples of SI-NET patients at different stages of disease were included in the study. The novel Ma2 autoantibody ELISA showed high sensitivity, specificity and accuracy with ROC curve analysis underlying an area between 0.734 and 0.816. Ma2 autoantibodies in the blood from SI-NET patients were verified by western blot and sequential immunoprecipitation. Serum antibodies of patients stain Ma2 in the tumor tissue and neurons. We observed that SI-NET patients expressing Ma2 autoantibody levels below the cutoff had a longer progression and recurrence-free survival compared to those with higher titer. We also detected higher levels of Ma2 autoantibodies in blood samples from TLC and ALC patients than from healthy controls, as previously shown in small cell lung carcinoma samples. Conclusion Here we show that high Ma2 autoantibody titer in the blood of SI-NET patients is a sensitive and specific biomarker, superior to chromogranin A (CgA) for the risk of recurrence after radical operation of these tumors.

A Novel Chromogranin-A Promoter-Driven Oncolytic Adenovirus for Midgut Carcinoid Therapy

Purpose: The use of replication-selective oncolytic adenoviruses is an emerging therapeutic approach for cancer, which thus far has not been employed for carcinoids. We therefore constructed Ad[CgA-E1A], a novel replication-selective oncolytic adenovirus, where the chromogranin A (CgA) promoter controls expression of the adenoviral E1A gene. Experimental Design: The Ad[CgA-E1A] virus was evaluated for E1A protein expression, replication ability, and cytolytic activity in various cell lines. It was also evaluated for treatment of xenografted human carcinoid tumors in nude mice. To use Ad[CgA-E1A] for the treatment of carcinoid liver metastases, it is important that normal hepatocytes do not support virus replication to minimize hepatotoxicity. We therefore evaluated CgA protein expression in normal hepatocytes. We also evaluated CgA gene expression in normal hepatocytes and microdissected tumor cells from carcinoid metastases. Results: We found that Ad[CgA-E1A] replicates similarly to wild-type virus in tumor cells with neuroendocrine features, including the BON carcinoid cell line and the SH-SY-5Y neuroblastoma cell lines, whereas it is attenuated in other cell types. Thus, cells where the CgA promoter is active are selectively killed. We also found that Ad[CgA-E1A] is able to suppress fast-growing human BON carcinoid tumors in nude mice. Furthermore, CgA is highly expressed in microdissected cells from carcinoid metastases, whereas it is not expressed in normal hepatocytes. Conclusion: Ad[CgA-E1A] is an interesting agent for the treatment of carcinoid liver metastases in conjunction with standard therapy for these malignancies.

Gene expression in midgut carcinoid tumors: potential targets for immunotherapy.

Classical midgut carcinoids are serotonin-secreting tumors derived from enterochromaffin cells in the gut. Metastatic disease represents a therapeutic challenge and immunotherapy implies a novel approach for treatment. In order to define antigens suitable for T-cell therapy with a preferential expression in midgut carcinoid tissue a broad screening of genes with preferential neuroendocrine restriction, genes described as over-expressed in various malignancies, and genes encoding cancer-testis associated antigens was performed. The expression of 32 genes was analyzed by reverse transcription polymerase chain reaction (RT-PCR) in 28 midgut carcinoid specimens, in the cell line BON and in normal tissues. Immunohistochemistry (IHC) was used to evaluate protein expression. Expression is shown of genes that have previously not been observed in midgut carcinoid tumors, such as Survivin and GAGEs. Also the expression is confirmed of genes that encode pivotal proteins in enterochromaffin cells, such as TPH1 and VMAT1, and their tissue-restricted expression is indicated. In addition, gene expression of IA-2 and CDX-2 in normal gastrointestinal (GI) tract and in tumor is shown. Protein expression of TPH, VMAT1, and Survivin was detected in tumor tissue. This study elucidates that TPH1, VMAT1, and Survivin should be further investigated as potential target antigens for T cell-mediated immunotherapy of midgut carcinoids.

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackie-adenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3–4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systemic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.

The somatostatin analogue octreotide inhibits growth of small intestine neuroendocrine tumour cells.

Octreotide is a widely used synthetic somatostatin analogue that significantly improves the management of neuroendocrine tumours (NETs). Octreotide acts through somatostatin receptors (SSTRs). However, the molecular mechanisms leading to successful disease control or symptom management, especially when SSTRs levels are low, are largely unknown. We provide novel insights into how octreotide controls NET cells. CNDT2.5 cells were treated from 1 day up to 16 months with octreotide and then were profiled using Affymetrix microarray analysis. Quantitative real-time PCR and western blot analyses were used to validate microarray profiling in silico data. WST-1 cell proliferation assay was applied to evaluate cell growth of CNDT2.5 cells in the presence or absence of 1 µM octreotide at different time points. Moreover, laser capture microdissected tumour cells and paraffin embedded tissue slides from SI-NETs at different stages of disease were used to identify transcriptional and translational expression. Microarrays analyses did not reveal relevant changes in SSTR expression levels. Unexpectedly, six novel genes were found to be upregulated by octreotide: annexin A1 (ANXA1), rho GTPase-activating protein 18 (ARHGAP18), epithelial membrane protein 1 (EMP1), growth/differentiation factor 15 (GDF15), TGF-beta type II receptor (TGFBR2) and tumour necrosis factor (ligand) superfamily member 15 (TNFSF15). Furthermore, these novel genes were expressed in tumour tissues at transcript and protein levels. We suggest that octreotide may use a potential novel framework to exert its beneficial effect as a drug and to convey its action on neuroendocrine cells. Thus, six novel genes may regulate cell growth and differentiation in normal and tumour neuroendocrine cells and have a role in a novel octreotide mechanism system.

CD8 + T cells against multiple tumor-associated antigens in peripheral blood of midgut carcinoid patients

PurposeThe aim of the study was to identify immunogenic HLA-A*0201-binding epitopes derived from a number of classical midgut carcinoid-associated proteins. CD8+ T cells recognizing tumor-associated antigen (TAA) epitopes are of great interest for the establishment of immunotherapy as a novel treatment for this type of malignancy.Experimental designMidgut carcinoid tumor specimens were microdissected and expression levels of potential TAAs were investigated by quantitative real time PCR. HLA-A*0201-binding motifs were selected using HLA peptide binding prediction algorithms and stabilization of HLA-A*0201 was verified using TAP-deficient T2 cells. Peripheral blood of midgut carcinoid patients was analyzed for peptide epitope recognition and the feasibility of generating peptide-reactive CD8+ T cells in healthy blood donors was examined by an in vitro stimulation protocol using mature DCs. Activation of patient and healthy donor CD8+ T cells was analyzed by intracellular flow cytometry staining of interferon γ.ResultsChromogranin A (CGA), tryptophan hydroxylase 1 (TPH-1), vesicular monoamine transporter 1 (VMAT-1), caudal type homeobox transcription factor 2 (CDX-2), and islet autoantigen 2 (IA-2) are properly expressed by midgut carcinoid tumor cells, with CGA mRNA expressed to greatest level. Midgut carcinoid patients have increased frequencies of peripheral blood CD8+ T cells recognizing a pool of HLA-A*0201 peptides derived from these proteins compared to healthy age-matched individuals. Activated peptide-specific CD8+ T cells could also be generated in healthy blood donors by in vitro stimulation.ConclusionWe have identified a number of immunogenic midgut carcinoid-associated peptide epitopes recognized by CD8+ T cells. We show that midgut carcinoid patients display immune recognition of their tumors. Memory CD8+ T cells in patient blood are of great interest when pursuing an immunotherapeutic treatment strategy.