Abscopal effect of radiation on lung metastases of hepatocellular carcinoma: a case report

<p>Abstract</p> <p>Introduction</p> <p>The abscopal effect is the effect of radiation therapy at a site distant to the area of irradiation. This is not a common event and has not been clearly defined, resulting in few reported cases in the literature. We discuss this phenomenon in a patient with hepatocellular carcinoma.</p> <p>Case presentation</p> <p>A 63-year-old Japanese man underwent extended right hepatic lobectomy for hepatocellular carcinoma. During his follow-up examination, a single lung metastasis and a single mediastinal lymph node metastasis were found. Trans-catheter arterial embolization was initially attempted to treat the mediastinal tumor, however this approach failed to take effect and carried risks of spinal artery embolism. External-beam irradiation, with a dose of 2.25 Gy per fraction, was performed using an antero-posterior parallel-opposed technique (total dose, 60.75 Gy). A computed tomography scan performed one month after starting radiotherapy showed a remarkable reduction of the mediastinal lymph node metastasis. In addition to this, we observed spontaneous shrinking of the lung metastasis, which was located in the right lower lobe and out of the radiation field. No chemotherapy was given during the period. There has been no recurrence of either the lung metastasis or the mediastinal lymph node metastasis during a follow-up 10 years after the radiotherapy.</p> <p>Conclusion</p> <p>We observed a rare abscopal effect in a site distant from the area of irradiation. Irradiation of the mediastinum resulted in tumor mass regression in the untreated lung tumor.</p

An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

PURPOSE:Recent studies have shown that a new generation of synthetic agonist of Toll-like receptor (TLR) 9 consisting a 3'-3'-attached structure and a dCp7-deaza-dG dinucultodie shows more potent immunostimulatory effects in both mouse and human than conventional CpG oligonucleotides. Radiation therapy (RT) provides a source of tumor antigens that are released from dying, irradiated, tumor cells without causing systemic immunosuppression. We, therefore, examined effect of combining RT with a designer synthetic agonist of TLR9 on anti-tumoral immunity, primary tumor growth retardation and metastases in a murine model of lung cancer. METHODS:Grouped C57BL/6 and congenic B cell deficient mice (B(-/-)) bearing footpad 3LL tumors were treated with PBS, TLR9 agonist, control oligonucelotide, RT or the combination of RT and TLR9 agonist. Immune phenotype of splenocytes and serum IFN-γ and IL-10 levels were analyzed by FACS and ELISA, 24 h after treatment. Tumor growth, lung metastases and survival rate were monitored and tumor specific antibodies in serum and deposition in tumor tissue were measured by ELISA and immunofluorescence. RESULTS:TLR9 agonist expanded and activated B cells and plasmacytoid dendritic cells in wild-type mice and natural killer DCs (NKDCs) in B cell-deficient (B(-/-)) mice bearing ectopic Lewis lung adenocarcinoma (3LL). Combined RT with TLR9 agonist treatment inhibited 3LL tumor growth in both wild type and B(-/-) mice. A strong tumor-specific humoral immune response (titer: 1/3200) with deposition of mouse IgG auto-antibodies in tumor tissue were found in wildtype mice, whereas the number of tumor infiltrating NKDCs increased in B(-/-) mice following RT+ TLR9 agonist therapy. Furthermore, mice receiving combination therapy had fewer lung metastases and a higher survival than single treatment cohorts. CONCLUSIONS:Combination therapy with TLR9 agonist and RT induces systemic anti-tumoral humoral response, augments tumoral infiltration of NKDCs, reduces pulmonary metastases and improves survival in a murine model of 3LL cancer

Mda-9/Syntenin Is Expressed in Uveal Melanoma and Correlates with Metastatic Progression

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound–healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target

A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies

INTRODUCTION: The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy. METHODS: Blood samples were collected from 61 locally advanced breast cancers (36 HER2- and 25 HER2+) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8+ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data. RESULTS: The proportion of circulating immune effectors was similar in HER2+ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4+/CD8+ T cell ratio (with a prevalence of naïve and central memory CD8+ T cells) were observed in HER2- cases. Higher numbers of circulating CD8+ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2+ cases, together with a higher prevalence of intratumor CD8+ T cells. Serum cytokine profile of HER2+ patients was similar to that of controls, whereas HER2- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2+ cases (IL-2, IL-1β, IL-8, IL-6, IL-10). CONCLUSIONS: Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2−/− γc−/− mouse

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2−/−γc−/− mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2−/− γc−/− mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation

A Toll-Like Receptor 2 Pathway Regulates the Ppargc1a/b Metabolic Co-Activators in Mice with Staphylococcal aureus Sepsis

Activation of the host antibacterial defenses by the toll-like receptors (TLR) also selectively activates energy-sensing and metabolic pathways, but the mechanisms are poorly understood. This includes the metabolic and mitochondrial biogenesis master co-activators, Ppargc1a (PGC-1α) and Ppargc1b (PGC-1β) in Staphylococcus aureus (S. aureus) sepsis. The expression of these genes in the liver is markedly attenuated inTLR2−/− mice and markedly accentuated in TLR4−/− mice compared with wild type (WT) mice. We sought to explain this difference by using specific TLR-pathway knockout mice to test the hypothesis that these co-activator genes are directly regulated through TLR2 signaling. By comparing their responses to S. aureus with WT mice, we found that MyD88-deficient and MAL-deficient mice expressed hepatic Ppargc1a and Ppargc1b normally, but that neither gene was activated in TRAM-deficient mice. Ppargc1a/b activation did not require NF-kβ, but did require an interferon response factor (IRF), because neither gene was activated in IRF-3/7 double-knockout mice in sepsis, but both were activated normally in Unc93b1-deficient (3d) mice. Nuclear IRF-7 levels in TLR2−/− and TLR4−/− mice decreased and increased respectively post-inoculation and IRF-7 DNA-binding at the Ppargc1a promoter was demonstrated by chromatin immunoprecipitation. Also, a TLR2-TLR4-TRAM native hepatic protein complex was detected by immunoprecipitation within 6 h of S. aureus inoculation that could support MyD88-independent signaling to Ppargc1a/b. Overall, these findings disclose a novel MyD88-independent pathway in S. aureus sepsis that links TLR2 and TLR4 signaling in innate immunity to Ppargc1a/b gene regulation in a critical metabolic organ, the liver, by means of TRAM, TRIF, and IRF-7

Novel Mouse Xenograft Models Reveal a Critical Role of CD4+ T Cells in the Proliferation of EBV-Infected T and NK Cells

Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, ectopically infects T or NK cells to cause severe diseases of unknown pathogenesis, including chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). We developed xenograft models of CAEBV and EBV-HLH by transplanting patients' PBMC to immunodeficient mice of the NOD/Shi-scid/IL-2Rγnull strain. In these models, EBV-infected T, NK, or B cells proliferated systemically and reproduced histological characteristics of the two diseases. Analysis of the TCR repertoire expression revealed that identical predominant EBV-infected T-cell clones proliferated in patients and corresponding mice transplanted with their PBMC. Expression of the EBV nuclear antigen 1 (EBNA1), the latent membrane protein 1 (LMP1), and LMP2, but not EBNA2, in the engrafted cells is consistent with the latency II program of EBV gene expression known in CAEBV. High levels of human cytokines, including IL-8, IFN-γ, and RANTES, were detected in the peripheral blood of the model mice, mirroring hypercytokinemia characteristic to both CAEBV and EBV-HLH. Transplantation of individual immunophenotypic subsets isolated from patients' PBMC as well as that of various combinations of these subsets revealed a critical role of CD4+ T cells in the engraftment of EBV-infected T and NK cells. In accordance with this finding, in vivo depletion of CD4+ T cells by the administration of the OKT4 antibody following transplantation of PBMC prevented the engraftment of EBV-infected T and NK cells. This is the first report of animal models of CAEBV and EBV-HLH that are expected to be useful tools in the development of novel therapeutic strategies for the treatment of the diseases

Mesenchymal Stem Cells in Early Entry of Breast Cancer into Bone Marrow

BACKGROUND: An understanding of BC cell (BCC) entry into bone marrow (BM) at low tumor burden is limited when compared to highly metastatic events during heavy tumor burden. BCCs can achieve quiescence, without interfering with hematopoiesis. This occurs partly through the generation of gap junctions with BM stroma, located close to the endosteum. These events are partly mediated by the evolutionary conserved gene, Tac1. METHODOLOGY/PRINCIPAL FINDINGS: This study focuses on the role of mesenchymal stem cells (MSCs), Tac1, SDF-1 and CXCR4 in BCC entry into BM. The model is established in studies with low numbers of tumor cells, and focuses on cancer cells with low metastatic and invasion potential. This allowed us to recapitulate early event, and to study cancer cells with low invasive potential, even when they are part of larger numbers of highly metastatic cells. A novel migration assay showed a facilitating role of MSCs in BCC migration across BM endothelial cells. siRNA and ectopic expression studies showed a central role for Tac1 and secondary roles for SDF-1alpha and CXCR4. We also observed differences in the mechanisms between low invasive and highly metastatic cells. The in vitro studies were verified in xenogeneic mouse models that showed a preference for low invasive BCCs to BM, but comparable movement to lung and BM by highly metastatic BCCs. The expressions of Tac1 and production of SDF-1alpha were verified in primary BCCs from paired samples of BM aspirates and peripheral blood. CONCLUSIONS/SIGNIFICANCE: MSC facilitate BCC entry into BM, partly through Tac1-mediated regulation of SDF-1alpha and CXCR4. We propose a particular population of BCC with preference for BM could be isolated for characterization. This population might be the subset that enter BM at an early time period, and could be responsible for cancer resurgence and resistance to current therapies