Multimodal Therapy Approaches for NUT Carcinoma by Dual Combination of Oncolytic Virus Talimogene Laherparepvec with Small Molecule Inhibitors.

NUT (nuclear-protein-in-testis) carcinoma (NC) is a highly aggressive tumor disease. Given that current treatment regimens offer a median survival of six months only, it is likely that this type of tumor requires an extended multimodal treatment approach to improve prognosis. In an earlier case report, we could show that an oncolytic herpes simplex virus (T-VEC) is functional in NC patients. To identify further combination partners for T-VEC, we have investigated the anti-tumoral effects of T-VEC and five different small molecule inhibitors (SMIs) alone and in combination in human NC cell lines. Dual combinations were found to result in higher rates of tumor cell reductions when compared to the respective monotherapy as demonstrated by viability assays and real-time tumor cell growth monitoring. Interestingly, we found that the combination of T-VEC with SMIs resulted in both stronger and earlier reductions in the expression of c-Myc, a main driver of NC cell proliferation, when compared to T-VEC monotherapy. These results indicate the great potential of combinatorial therapies using oncolytic viruses and SMIs to control the highly aggressive behavior of NC cancers and probably will pave the way for innovative multimodal clinical studies in the near future.

In Vitro Sensitivity of Neuroendocrine Neoplasms to an Armed Oncolytic Measles Vaccine Virus.

Neuroendocrine neoplasms represent a heterogenous group of rare tumors whose current therapeutic options show only limited efficacy. Oncolytic viruses exert their mode of action through (onco-)lysis of infected tumor cells and the induction of a systemic antitumoral immune response in a virus-induced inflammatory micromilieu. Here, we investigated the potential of our well-established second-generation suicide-gene armed oncolytic measles vaccine virus (MeV-SCD) in five human NEN cell lines. First, (i) expression of the MeV receptor CD46 and (ii) its correlation with primary infection rates were analyzed. Next, (iii) promising combination partners for MeV-SCD were tested by employing either the prodrug 5-fluorocytosine, which is converted into the chemotherapeutic compound 5-fluorouracil, or the mTOR-inhibitor everolimus. As a result, MeV-SCD was found to kill all NEN tumor cell lines efficiently in a dose-dependent manner. This oncolytic effect was further enhanced by exploiting the prodrug-converting system, which was found to be highly instrumental in overcoming the partial resistance found in a single NEN cell line. Furthermore, viral replication was unaffected by everolimus, which is a basic requirement for combined use in NEN patients. These data suggest that MeV-SCD has profound potential for patients with NEN, thus paving the way for early clinical trials.

Efficacy of Different Oncolytic Vaccinia Virus Strains for the Treatment of Murine Peritoneal Mesothelioma.

Effective treatment options for peritoneal surface malignancies (PSMs) are scarce. Oncolytic virotherapy with recombinant vaccinia viruses might constitute a novel treatment option for PSM. We aimed to identify the most effective oncolytic vaccinia virus strain in two murine mesothelioma cell lines and the oncolytic potential in a murine model of peritoneal mesothelioma. Cell lines AB12 and AC29 were infected in vitro with vaccinia virus strains Lister (GLV-1h254), Western Reserve (GLV-0b347), and Copenhagen (GLV-4h463). The virus strain GLV-0b347 was shown most effective in vitro and was further investigated by intraperitoneal (i.p.) application to AB12 and AC29 mesothelioma-bearing mice. Feasibility, safety, and effectiveness of virotherapy were assessed by evaluating the peritoneal cancer index (PCI), virus detection in tumor tissues and ascites, virus growth curves, and comparison of overall survival. After i.p. injection of GLV-0b347, virus was detected in both tumor cells and ascites. In comparison to mock-treated mice, overall survival was significantly prolonged, ascites was less frequent and PCI values declined. However, effective treatment was only observed in animals with limited tumor burden at the time point of virus application. Nonetheless, intraperitoneal virotherapy with GLV-0b347 might constitute a novel therapeutic option for the treatment of peritoneal mesothelioma. Additional treatment modifications and combinational regimes will be investigated to further enhance treatment efficacy.

Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model.

Oncolytic virotherapy constitutes a promising treatment option for many solid cancers, including peritoneal carcinomatosis (PC), which still represents a terminal stage of many types of tumors. To date, the in vitro efficacy of oncolytic viruses is mostly tested in 2D-cultured tumor cell lines due to the lack of realistic 3D in vitro tumor models. We have investigated the feasibility of virotherapy as a treatment option for PC in a human ex vivo peritoneum co-culture model. Human HT-29 cancer cells stably expressing marker genes GFP and firefly luciferase (GFP/luc) were cultured on human peritoneum and infected with two prototypic oncolytic viruses (GLV-0b347 and MeV-DsRed). Both viral constructs were able to infect HT-29 cells in patient-derived peritoneum with high tumor specificity. Over time, both GFP signal and luciferase activity decreased substantially, thereby indicating successful virus-induced oncolysis. Furthermore, immunohistochemistry stainings showed specific virotherapeutic infections of HT-29 cells and effective tumor cell lysis in infected co-cultures. Thus, the PC model established here provides a clinically relevant screening platform to evaluate the therapeutic efficacy of virotherapeutic compounds and also to investigate, in an autologous setting, the immunostimulatory potential of oncolytic viruses for PC in a unique human model system superior to standard 2D in vitro models.

Case report: Immunovirotherapy as a novel add-on treatment in a patient with thoracic NUT carcinoma.

NUT carcinoma (NC) is a rare and extremely aggressive form of cancer, usually presenting with intrathoracic or neck manifestations in adolescents and young adults. With no established standard therapy regimen and a median overall survival of only 6.5 months, there is a huge need for innovative treatment options. As NC is genetically driven by a single aberrant fusion oncoprotein, it is generally characterized by a low tumor mutational burden, thus making it immunologically cold and insusceptible to conventional immunotherapy. Recently, we have demonstrated that oncolytic viruses (OVs) are able to specifically infect and lyse NC cells, thereby turning an immunologically cold tumor microenvironment into a hot one. Here, we report an intensive multimodal treatment approach employing for the first time an OV (talimogene laherparepvec (T-VEC); IMLYGIC®) together with the immune checkpoint inhibitor pembrolizumab as an add-on to a basic NC therapy (cytostatic chemotherapy, radiation therapy, epigenetic therapy) in a patient suffering from a large thoracic NC tumor which exhibits an aberrant, unique BRD3:NUTM1 fusion. This case demonstrates for the first time the feasibility of this innovative add-on immunovirotherapy regimen with a profound, repetitive and durable replication of T-VEC that is instrumental in achieving tumor stabilization and improvement in the patient´s quality of life. Further, a previously unknown BRD3:NUTM1 fusion gene was discovered that lacks the extraterminal domain of BRD3.

Oncolytic viruses: challenges and considerations in an evolving clinical landscape.

Despite advances in treatment, cancer remains a leading cause of death worldwide. Although treatment strategies are continually progressing, cancers have evolved many mechanisms for evading therapies and the host immune system. Oncolytic viruses (OVs) could provide a much-needed option for cancers that are resistant to existing treatments. OVs can be engineered to specifically target and kill cancer cells, while simultaneously triggering an immune response at the site of infection. This review will focus on the challenges of developing a successful OV and translation to clinical practice, discussing the innovative strategies that are being used to optimize the potential of OVs. Here, we will also explore the current clinical landscape and the prospects of OVs in early clinical development.

Oncolytic viruses (OVs) are viruses that may help destroy tumor cells. They work by selectively infecting and replicating within tumor cells, causing the cells to burst and release newly built viruses. These viruses infect nearby tumor cells, triggering the body's immune system to attack the tumor and any tumor cells that have spread throughout the body. Clinical trials have shown that OVs can destroy cancer cells that are resistant to standard therapies. OVs in combination with other cancer therapies can be more effective and there are over 100 clinical trials planned, ongoing or completed to investigate this approach. OVs are generally well tolerated, the most common treatment-related side effects include fever, aches and pains, and tiredness for 1­2 days. While only four OVs have been approved so far, there are more expected to come. Overall, OVs may provide a way to directly destroy tumors and turn on the immune system to destroy tumor cells throughout the body.

Efficacy of Oncolytic Herpes Simplex Virus T-VEC Combined with BET Inhibitors as an Innovative Therapy Approach for NUT Carcinoma.

NUT carcinoma (NC) is an extremely aggressive tumor and current treatment regimens offer patients a median survival of six months only. This article reports on the first in vitro studies using immunovirotherapy as a promising therapy option for NC and its feasible combination with BET inhibitors (iBET). Using NC cell lines harboring the BRD4-NUT fusion protein, the cytotoxicity of oncolytic virus talimogene laherparepvec (T-VEC) and the iBET compounds BI894999 and GSK525762 were assessed in vitro in monotherapeutic and combinatorial approaches. Viral replication, marker gene expression, cell proliferation, and IFN-ß dependence of T-VEC efficiency were monitored. T-VEC efficiently infected and replicated in NC cell lines and showed strong cytotoxic effects. This implication could be enhanced by iBET treatment following viral infection. Viral replication was not impaired by iBET treatment. In addition, it was shown that pretreatment of NC cells with IFN-ß does impede the replication as well as the cytotoxicity of T-VEC. T-VEC was found to show great potential for patients suffering from NC. Of note, when applied in combination with iBETs, a reinforcing influence was observed, leading to an even stronger anti-tumor effect. These findings suggest combining virotherapy with diverse molecular therapeutics for the treatment of NC.

Virotherapy in Germany-Recent Activities in Virus Engineering, Preclinical Development, and Clinical Studies.

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review-as part of the special edition on "State-of-the-Art Viral Vector Gene Therapy in Germany"-the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology.

Assessing and Overcoming Resistance Phenomena against a Genetically Modified Vaccinia Virus in Selected Cancer Cell Lines.

Genetically modified vaccinia viruses (VACVs) have been shown to possess profound oncolytic capabilities. However, tumor cell resistance to VACVs may endanger broad clinical success. Using cell mass assays, viral replication studies, and fluorescence microscopy, we investigated primary resistance phenomena of cell lines of the NCI-60 tumor cell panel to GLV-1h94, a derivative of the Lister strain of VACV, which encodes the enzyme super cytosine deaminase (SCD) that converts the prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU). After treatment with GLV-1h94 alone, only half of the cell lines were defined as highly susceptible to GLV-1h94-induced oncolysis. When adding 5-FC, 85% of the cell lines became highly susceptible to combinatorial treatment; none of the tested tumor cell lines exhibited a "high-grade resistance" pattern. Detailed investigation of the SCD prodrug system suggested that the cytotoxic effect of converted 5-FU is directed either against the cells or against the virus particles, depending on the balance between cell line-specific susceptibility to GLV-1h94-induced oncolysis and 5-FU sensitivity. The data provided by this work underline that cellular resistance against VACV-based virotherapy can be overcome by virus-encoded prodrug systems. Phase I/II clinical trials are recommended to further elucidate the enormous potential of this combination therapy.

Oncolytic vaccinia virus GLV-1h68 exhibits profound antitumoral activities in cell lines originating from neuroendocrine neoplasms.

BACKGROUND: Oncolytic virotherapy is an upcoming treatment option for many tumor entities. But so far, a first oncolytic virus only was approved for advanced stages of malignant melanomas. Neuroendocrine tumors (NETs) constitute a heterogenous group of tumors arising from the neuroendocrine system at diverse anatomic sites. Due to often slow growth rates and (in most cases) endocrine non-functionality, NETs are often detected only in a progressed metastatic situation, where therapy options are still severely limited. So far, immunotherapies and especially immunovirotherapies are not established as novel treatment modalities for NETs. METHODS: In this immunovirotherapy study, pancreatic NET (BON-1, QGP-1), lung NET (H727, UMC-11), as well as neuroendocrine carcinoma (NEC) cell lines (HROC-57, NEC-DUE1) were employed. The well characterized genetically engineered vaccinia virus GLV-1 h68, which has already been investigated in various clinical trials, was chosen as virotherapeutical treatment modality. RESULTS: Profound oncolytic efficiencies were found for NET/NEC tumor cells. Besides, NET/NEC tumor cell bound expression of GLV-1 h68-encoded marker genes was observed also. Furthermore, a highly efficient production of viral progenies was detected by sequential virus quantifications. Moreover, the mTOR inhibitor everolimus, licensed for treatment of metastatic NETs, was not found to interfere with GLV-1 h68 replication, making a combinatorial treatment of both feasible. CONCLUSIONS: In summary, the oncolytic vaccinia virus GLV-1 h68 was found to exhibit promising antitumoral activities, replication capacities and a potential for future combinatorial approaches in cell lines originating from neuroendocrine neoplasms. Based on these preliminary findings, virotherapeutic effects now have to be further evaluated in animal models for treatment of Neuroendocrine neoplasms (NENs).

Biological treatment of pediatric sarcomas by combined virotherapy and NK cell therapy.

BACKGROUND: In pediatric sarcomas, outcomes of established therapies still remain poor, especially due to high-grade resistances to chemotherapeutic compounds. Taking novel biological approaches into account, virotherapy was found to be efficient in many pediatric sarcoma types. Also NK cell therapy was denoted to represent a promising upcoming strategy for pediatric sarcoma patients. We here investigated a combinatorial approach employing oncolytic measles vaccine virotherapeutics (MeV) together with activated human NK cells (or PBMCs). METHODS: The human sarcoma cell lines A673 and HT1080 were used to evaluate the efficacy of this combinatorial treatment modality. Oncolysis was determined by measuring real-time cell proliferation using the xCELLigence RTCA SP system. Furthermore, expression of receptors on NK cells and the respective ligands on A673 cells was analyzed by flow cytometry. To measure the protein release of activated NK cells a LEGENDplex™ assay was performed. RESULTS: Monotherapy with MeV led to a time- and dose-dependent oncolytic reduction of A673 and HT1080 sarcoma tumor cell masses. Concurrently, such MeV infections did not change the expression of NK cell ligands MICA/B, ULBP1, 2, and 3, CD112, and CD155. As shown by real-time proliferation assays, infections of A673 and HT1080 sarcoma cells with MeV followed by co-culture with activated NK cells or PBMCs led to enhanced sarcoma cell destruction when compared to the respective monotherapies. In parallel, this dual therapy resulted in an increased release of granzymes, perforin, and granulysin from NK cells. In contrast, expression of activation and ontogenesis receptors on NK cells was not found to be altered after co-culture with MeV-infected A673 sarcoma cells. CONCLUSIONS: Taken together, the combined treatment strategy comprising oncolytic MeV and activated NK cells resulted in enhanced oncolysis of A673 and HT1080 cells when compared to the respective monotherapies. In parallel, we observed an increased release of NK cell activation markers upon co-culture with MeV-infected A673 human sarcoma cells. These results support the onset of clinical trials combining oncolytic virotherapy with NK cell based immunotherapies.

Starvation-Induced Differential Virotherapy Using an Oncolytic Measles Vaccine Virus.

Starvation sensitizes tumor cells to chemotherapy while protecting normal cells at the same time, a phenomenon defined as differential stress resistance. In this study, we analyzed if starvation would also increase the oncolytic potential of an oncolytic measles vaccine virus (MeV-GFP) while protecting normal cells against off-target lysis. Human colorectal carcinoma (CRC) cell lines as well as human normal colon cell lines were subjected to various starvation regimes and infected with MeV-GFP. The applied fasting regimes were either short-term (24 h pre-infection) or long-term (24 h pre- plus 96 h post-infection). Cell-killing features of (i) virotherapy, (ii) starvation, as well as (iii) the combination of both were analyzed by cell viability assays and virus growth curves. Remarkably, while long-term low-serum, standard glucose starvation potentiated the efficacy of MeV-mediated cell killing in CRC cells, it was found to be decreased in normal colon cells. Interestingly, viral replication of MeV-GFP in CRC cells was decreased in long-term-starved cells and increased after short-term low-glucose, low-serum starvation. In conclusion, starvation-based virotherapy has the potential to differentially enhance MeV-mediated oncolysis in the context of CRC cancer patients while protecting normal colon cells from unwanted off-target effects.

Phase I Study of Oncolytic Vaccinia Virus GL-ONC1 in Patients with Peritoneal Carcinomatosis.

Purpose: Peritoneal carcinomatosis is common in advanced tumor stages or disease recurrence arising from gastrointestinal cancers, gynecologic malignancies, or primary peritoneal carcinoma. Because current therapies are mostly ineffective, new therapeutic approaches are needed. Here, we report on a phase I study designed to assess safety, MTD, and antitumor activity of intraperitoneal administration of oncolytic vaccinia virus GL-ONC1 in advanced stage peritoneal carcinomatosis patients.Patients and Methods: GL-ONC1 was administered intraperitoneally every 4 weeks for up to four cycles at three different dose levels (107-109 pfu) following a standard 3+3 dose escalation design. GL-ONC1 was infused via an indwelling catheter that enabled repetitive analyses of peritoneal fluid biopsies. The primary study objective was safety of GL-ONC1 according to Common Terminology Criteria for Adverse Events, version 4.0 (CTCAEv4.0).Results: Patients with advanced-stage peritoneal carcinomatosis (n = 7) or advanced peritoneal mesothelioma (n = 2) received 24 doses of GL-ONC1. Adverse events were limited to grades 1-3, including transient flu-like symptoms and increased abdominal pain, resulting from treatment-induced peritonitis. No DLT was reported, and the MTD was not reached. Furthermore, no signs of viral shedding were observed. Importantly, in 8 of 9 study patients, effective intraperitoneal infections, in-patient replication of GL-ONC1, and subsequent oncolysis were demonstrated in cycle 1. All patients developed neutralizing activities against GL-ONC1.Conclusions: GL-ONC1 was well tolerated when administered into the peritoneal cavity of patients with advanced stage peritoneal carcinomatosis. Efficient tumor cell infection, in-patient virus replication, and oncolysis were limited to treatment cycle 1 (ClinicalTrials.gov number, NCT01443260). Clin Cancer Res; 24(18); 4388-98. ©2018 AACR.

Chemovirotherapy of Pancreatic Adenocarcinoma by Combining Oncolytic Vaccinia Virus GLV-1h68 with nab-Paclitaxel Plus Gemcitabine.

Oncolytic viruses have proven their therapeutic potential against a variety of different tumor entities both in vitro and in vivo. Their ability to selectively infect and lyse tumor cells, while sparing healthy tissues, makes them favorable agents for tumor-specific treatment approaches. Particularly, the addition of virotherapeutics to already established chemotherapy protocols (so-called chemovirotherapy) is of major interest. Here we investigated the in vitro cytotoxic effect of the oncolytic vaccinia virus GLV-1h68 combined with dual chemotherapy with nab-paclitaxel plus gemcitabine in four human pancreatic adenocarcinoma cell lines (AsPc-1, BxPc-3, MIA-PaCa-2, and Panc-1). This chemovirotherapeutic protocol resulted in enhanced tumor cell killing in two tumor cell lines compared to the respective monotherapies. We were thereby able to show that the combination of oncolytic vaccinia virus GLV-1h68 with nab-paclitaxel and gemcitabine has great potential in the chemovirotherapeutic treatment of advanced pancreatic adenocarcinoma. However, the key to a successful combinatorial chemovirotherapeutic treatment seems to be a profound viral replication, as tumor cell lines that were non-responsive to the combination therapy exhibited a reduced viral replication in the presence of the chemotherapeutics. This finding is of special significance when aiming to achieve a virus-mediated induction of a profound and long-lasting antitumor immunity.

The impact of family environment on the development of alcohol dependence.

BACKGROUND: Alcoholism is a family disease. Many studies confirm that a family history of alcoholism is associated with the development of later alcohol dependence. The aim of this study is to analyze the impact of family structure and relations between its members in the development of alcohol addiction in children grown up in these families. SUBJECTS AND METHODS: The research study was based on authors` anonymous questionnaire including questions referring to: family structure, parents' divorce, prevalence of alcoholism in the family, parents' attitude towards alcohol and parent-child relationships. The study group consisted of 125 people, 83 men and 42 women, aged from 22 to 68 participating in treatment programs for alcohol addiction. The control group consisted of 231 people, 136 men and 95 women, age from 17 to 65, with no history of alcoholism. RESULTS: The study group participants stated less frequently that they had been raised by both parents (78% vs 87%, p<0.05). In this group one of the parents significantly more frequently abused alcohol (43% vs 19%; p<0.05) or both parents abused alcohol (15% vs 1%; p<0.05). The participants also claimed to be more often punished for their failures, abused physically/verbally and could less often depend on their parents. CONCLUSIONS: Based on these results we concluded that patients addicted to alcohol were more often raised by a single parent, they were more likely to have alcohol-dependent parents and relationships with their parents were more often impaired.

Association of early drinking onset with subsequent alcohol abuse.

BACKGROUND: In the recent years the phenomenon of early alcohol initiation is observed. This problem is often underestimated, in spite of its numerous negative consequences SUBJECTS AND METHODS: The research study was based on authors` anonymous questionnaire including questions referring to: age of alcohol initiation, age of the first blackout after drinking alcohol, the place and circumstances of alcohol initiation and the reason of drinking alcohol for the first time. The study group consisted of 125 people, 83 men and 42 women, aged from 22 to 68 participating in treatment programs for alcohol addiction. RESULTS: In the study group it occurred before the age of 15 more often than in the control group (49% vs. 42%). The same correlation exists for the alcohol initiation before 12 years of age (13% vs. 8%) and is statistically significant (p<0.05). What's very alarming drinking alcohol for the first time took place for some of the respondents before the age of 10 and also significantly more often in the study group (6% vs. 2%, p<0.05). CONCLUSIONS: The obtained results allow to conclude that in patients addicted to alcohol the initiation took place earlier than in the study group (age 13-15 vs. 16-18). Also, very early alcohol initiation (<12 years) occurred more frequently in the study group (12.8% vs 8.2%). Based on our research, we confirmed that early drinking onset is associated with subsequent alcohol dependence.