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Background:
Recent decades have seen a rise in the incidence of well-differentiated (mainly papillary) thyroid carcinoma around the world. In Germany, the age-adjusted incidence of well-differentiated thyroid carcinoma in 2010 was 3.5 per 100 000 men and 8.7 per 100 000 women per year.
Method:
This review is based on randomized, controlled trials and multicenter trials on the treatment of well-differentiated thyroid carcinoma that were retrieved by a selective literature search, as well as on three updated guidelines issued in the past two years.
Results:
The recommended extent of surgical resection depends on whether the tumor is classified as low-risk or high-risk, so that papillary microcar cinomas, which carry a highly favorable prognosis, will not be overtreated. More than 90% of localized, well-differentiated thyroid carcinomas can be cured with a combination of surgery and radioactive iodine therapy. Radio active iodine therapy is also effective in the treatment of well-differentiated thyroid carcinomas with distant metastases, yielding a 10-year survival rate of 90%, as long as there is good iodine uptake and the tumor goes into remission after treatment; otherwise, the 10-year survival rate is only 10%. In the past two years, better treatment options have become available for radioactive-iodine-resistant thyroid carcinoma. Phase 3 studies of two different tyrosine kinase inhibitors have shown that either one can markedly prolong progression-free survival, but not overall survival. Their more common clinically significant side effects are hand-foot syndrome, hypertension, diarrhea, proteinuria, and weight loss.
Conclusion:
Slow tumor growth, good resectability, and susceptibility to radioactive iodine therapy lend a favorable prognosis to most cases of well-differentiated thyroid carcinoma. The treatment should be risk-adjusted and interdisciplinary, in accordance with the current treatment guidelines. Even metastatic thyroid carcinoma has a favorable prognosis as long as there is good iodine uptake. The newly available medical treatment options for radioactive-iodine-resistant disease need to be further studied.
A promising new approach for the treatment of human cancer is the use of oncolytic viruses, which exhibit tumor tropism. One of the top candidates in this area is the oncolytic vaccinia virus (VACV), which has already shown promising results in animal studies and in clinical trials. However, due to discrepancies in both innate and adaptive immunity between mice and men the evaluation of the vaccinia virus’ interactions with the host immune system in mice are not fully conclusive of what is actually happening in human cancer patients after systemic administration of vaccinia virus. Also, ethical and legal concerns as well as risk of potential toxicity limit research involving human patients. Therefore, a good in vivo model for testing interactions between vaccinia virus and human immune cells, avoiding the numerous limitations and risks associated with human studies, could be a humanized mouse model.
LIVP-1.1.1, GLV-2b372, GLV-1h68, GLV-1h375, GLV-1h376 and GLV-1h377 VACVs were provided by Genelux Corporation. GLV-2b372 was constructed by inserting TurboFP635 expression cassette into the J2R locus of the parental LIVP-1.1.1. GLV-1h375, -1h376 and -1h377 VACVs encode the human CTLA4-blocking single-chain antibody (CTLA4 scAb). Performed replication and cytotoxicity assays demonstrated that all six viruses were able to infect, replicate in and kill human tumor cells in virus-dose- and time-dependent fashion. CTLA4 scAb and β-glucuronidase (GusA) expression as well as viral titers in GLV-1h376-infected cells were analyzed by ELISA, β-glucuronidase assay and standard plaque assay, respectively, and compared. An excellent correlation with correlation coefficients R2>0.9806 were observed. GLV-1h376-encoded CTLA4 scAb was successfully purified from supernatants of infected CV-1 cells and demonstrated in vitro affinity to its human CTLA4 target and lack of cross-reactivity to mouse CTLA4. CTLA4 scAb functionality was confirmed in Jurkat cells. LIVP-1.1.1, GLV-2b372, GLV-1h68 and GLV-1h376 were next studied in non-tumorous and/or tumor-bearing humanized mice.
It was demonstrated that injection of human CD34+ stem cells into the liver of preconditioned newborn NSG mice let to a successful systemic reconstitution with human immune cells. CD19+ B cells, CD4 and CD8 single positive CD3+ T cell, NKp46+CD56- and NKp46+CD56+ NK cells as well as CD33+ myeloid cells developed. At early time points after engraftment, majority of the human hematopoietic cells detected in the mouse blood were CD19+ B cells and only a small portion were CD3+ T cells. With time a significant change in CD19+/CD3+ ratio was reported with a decrease of B cells and an increase of T cells. Implantation of A549 cells under the skin of those humanized NSG mice resulted in a progressive tumor growth, described for the first time in this thesis. Successful colonization of subcutaneous A549 tumors with VACVs was visualized and demonstrated by detection of virus-mediated TurboFP635 and GFP expression as well as by standard plaque assay and immunohistochemistry. The human CD45+ cell population in tumors was represented mainly by NKp46+CD56bright NK cells and a large portion of activated CD4+ and cytotoxic CD8+ T cells. However, no significant differences were observed between control and LIVP-1.1.1-infected tumors, suggesting that the recruitment of NK and activated T cells were more tumor tissue specific than virus-dependent. Unfortunately, virus-mediated CTLA4 scAb expression in the GLV-1h376-infected tumors was also not able to significantly increase activation of T cells compared to control and GLV-1h68-treated mice. Importantly, ELISA, β-glucuronidase and standard plaque assays showed an excellent correlation with correlation coefficients R2>0.9454 between CTLA4 scAb, GusA concentrations and viral titers in tumor samples from those GLV-1h376 treated mice.
T cells isolated from the spleens of such control or GLV-1h68- or -1h376-treated A549 tumor-bearing mice were functional and could successfully be activated with human T cells activation beads. However, although no significant difference was observed between the three mouse groups, a slightly higher percentage of the GLV-1h376-treated mice-derived T cells were expressing CD25 and producing IFN-ɣ after ex vivo activation, probably due to the CTLA4 blockade by the virus-encoded CTLA4 scAb in the GLV-1h376-treated mice. Also, slightly higher levels of IL-2 were detected in the culture supernatant of those splenocytes compared to control samples. In contrast, T cells from all three mouse groups were not able be activated by A549 tumor cells ex vivo.
Our model has the specific advantage that tumors develop under the skin of the humanized mice, which allows accurate monitoring of the tumor growth and evaluation of the oncolytic virotherapy. Therefore it is important to choose the right approaches for its further improvement.
Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV). Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4%) MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients.
Background
Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors.
Methods
For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation.
Results
GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV.
Conclusions
Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.
The recently discovered human DREAM complex (for DP, RB-like, E2F and MuvB complex) is a chromatin-associated pocket protein complex involved in cell cycle- dependent gene expression. DREAM consists of five core subunits and forms a complex either with the pocket protein p130 and the transcription factor E2F4 to repress gene expression or with the transcription factors B-MYB and FOXM1 to promote gene expression.
Gas2l3 was recently identified by our group as a novel DREAM target gene. Subsequent characterization in human cell lines revealed that GAS2L3 is a microtubule and F-actin cross-linking protein, expressed in G2/M, plays a role in cytokinesis, and is important for chromosomal stability.
The aim of the first part of the study was to analyze how expression of GAS2L3 is regulated by DREAM and to provide a better understanding of the function of GAS2L3 in mitosis and cytokinesis.
ChIP assays revealed that the repressive and the activating form of DREAM bind to the GAS2L3 promoter. RNA interference (RNAi) mediated GAS2L3 depletion demonstrated the requirement of GAS2L3 for proper cleavage furrow ingression in cytokinesis. Immunofluorescence-based localization studies showed a localization of GAS2L3 at the mitotic spindle in mitosis and at the midbody in cytokinesis. Additional experiments demonstrated that the GAS2L3 GAR domain, a putative microtubule- binding domain, is responsible for GAS2L3 localization to the constriction zones in cytokinesis suggesting a function for GAS2L3 in the abscission process.
DREAM is known to promote G2/M gene expression. DREAM target genes include several mitotic kinesins and mitotic microtubule-associated proteins (mitotic MAPs). However, it is not clear to what extent DREAM regulates mitotic kinesins and MAPs, so far. Furthermore, a comprehensive study of mitotic kinesin expression in cancer cell lines is still missing.
Therefore, the second major aim of the thesis was to characterize the regulation of mitotic kinesins and MAPs by DREAM, to investigate the expression of mitotic kinesins in cancer cell line panels and to evaluate them as possible anti-cancer targets.
ChIP assays together with RNAi mediated DREAM subunit depletion experiments demonstrated that DREAM is a master regulator of mitotic kinesins. Furthermore, expression analyses in a panel of breast and lung cancer cell lines revealed that mitotic kinesins are up-regulated in the majority of cancer cell lines in contrast to non-transformed controls. Finally, an inducible lentiviral-based shRNA system was developed to effectively deplete mitotic kinesins. Depletion of selected mitotic kinesins resulted in cytokinesis failures and strong anti-proliferative effects in several human cancer cell lines.
Thus, this system will provide a robust tool for future investigation of mitotic kinesin function in cancer cells.
Objective: The situation of patients with multiple myeloma, whose treatment often implies high-dose chemotherapy and stem cell transplantation that can be associated with severe symptoms and psychological distress, has gained attention in recent psychooncological research. This study followed an idiographic approach in order to identify the areas of life most relevant for the interviewed myeloma patients’ quality of life (QoL) as well as their current satisfaction with these.
Methods: 64 patients took part in semi-structured interviews according to the SEIQoL-DW Manual (Schedule for the Evaluation of Individual Quality of Life – Direct Weighting). Visual analogue scales (VAS) were used to gain additional information about a general assessment of the present QoL. Qualitative data evaluation preceded quantitative processing. Groups were compared according to the time elapsed since diagnosis regarding specified areas of life, satisfaction with these and their relative weighting. SEIQoL-DW-indices were correlated to the VAS to reflect on an interindividually comparable parameter.
Results: Personal social relationships were mentioned significantly more often as important for QoL than healthrelated aspects, and in direct comparison were weighted significantly stronger. Regarding the change of areas relevant for QoL over the time elapsed since diagnosis, there was a significant difference between groups concerning the area of spirituality. Satisfaction differed significantly between groups for the field of leisure.
Conclusion: The results for the interviewed patients with multiple myeloma point out the need to take into account the importance of social and individual aspects when reflecting on QoL. Similar findings have been reported for different samples. The relevance of an individualized approach is illustrated by the fact that individually named areas of life were rated comparatively strongly in their importance for the patients’ QoL. An overall assessment for the current QoL by means of VAS is regarded as an adequate supplement to the SEIQoL-Profile and an alternative to the SEIQoL-DW-Index.
Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers.
Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models.
Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke’s type A-stage HCT-116 and Duke’s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.
Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.
CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology ( 5 transmembrane domains) and a deviating spectrum of functions can be expected.