2604 Background: Oncolytic viruses can convert poorly immunogenic tumors into inflamed, immune-responsive lesions, but clinical benefit is often limited by insufficient induction of tumor antigen–specific T cells. PeptiCRAd-1 is a novel peptide-guided oncolytic adenoviral immunotherapy designed to deliver tumor antigens while providing intrinsic immune stimulation. It consists of a conditionally replicating adenovirus expressing CD40L and OX40L and coated with NY-ESO-1 and MAGE-A3 peptides to enhance priming of antigen-specific adaptive immunity. Methods: VALO-001 (NCT05492682) is an ongoing, open-label, non-randomized phase 1 trial enrolling adults with injectable advanced solid tumors expressing NY-ESO-1 and/or MAGE-A3, including melanoma, TNBC, NSCLC, sarcomas, and colorectal cancer. Treatment includes low-dose cyclophosphamide followed by intratumoral PeptiCRAd-1 prime/boost injections combined with pembrolizumab. Primary objectives are safety and tolerability; secondary/exploratory endpoints assess innate and adaptive immune activation: antigen-specific T-cell induction, tumor-infiltrating lymphocyte (TIL) density, viral shedding, and preliminary antitumor activity. Immune monitoring includes cytokine profiling, ELISpot for NY-ESO-1/MAGE-A3 responses, and paired biopsies for multiparametric immunophenotyping. Results: Seven patients have been enrolled and treated in the START Phase 1 evaluation of PeptiCRAd-1: five in the intratumoral (i.t.) cohort and two in the combined intratumoral plus subcutaneous (i.t. + s.c.) cohort. Across both groups, study treatment has been feasible and well tolerated, with no dose-limiting toxicities, treatment-related serious adverse events, or unexpected safety signals reported to date. In the i.t. cohort, innate immune activation occurred in 3/5 patients; NY-ESO-1/MAGE-A3-specific T-cell responses in 3/5; and increased TILs in 4/5. Clinical responses were observed in 2/5, with disease control in 3/5 patients. Early i.t. + s.c. data show markedly stronger peptide-specific T-cell responses than i.t. alone, supporting combined local–systemic immunization. This cohort remains open, with eight additional patients required. Across evaluable patients, biopsies demonstrated increased TIL density, and early antitumor activity—including partial responses and durable stable disease—was observed across multiple tumor types. Conclusions: PeptiCRAd-1 shows a favorable safety profile and induces coordinated systemic and intratumoral immune activation in advanced solid tumors. Robust antigen-specific T-cell responses and enhanced TIL infiltration support its potential to strengthen antitumor immunity and justify continued evaluation, including combination with PD-1 blockade. Clinical trial information: NCT05492682 .

Targeted cell entry with lentiviral vectors (LVs) is one approach to enable efficient and specific gene delivery in the context of immunotherapies, such as chimeric antigen receptor (CAR)-T cell therapy. We have recently shown the generation of CAR-T products with varying CD4:CD8 ratios with our selective, universal Adapter-LV system. However, low viral vector titers limit the clinical and in vivo applications of our measles virus (MV)-based system. Here, we report a novel pseudotype based on canine distemper virus (CDV) for the Adapter-LV system (CDV-Ad-LV) and show improved viral vector yield while maintaining flexibility, selectivity, and transduction efficacy. As for MV, the engineered CDV envelope protein is fused to a single-chain variable fragment that binds biotin in the context of a defined chemical linker. Thereby, Adapter-LVs bind only to biotinylated adapter molecules, which in turn bind the antigen of choice. Besides extensive characterization in vitro, we evaluated transduction in vivo with CDV-Ad-LVs and showed high transduction efficiencies of CD4+ and CD8+ T cells following a single-dose injection into human PBMC-transplanted immunodeficient non-obese diabetic (NOD) SCID gamma (NSG) mice. Hence, CDV-Ad-LVs represent a novel and efficient system for adapter-mediated transduction, which broadens the potential of the Adapter-LV technology, going beyond CAR-T cell therapy applications.

Rare cancers present significant challenges in diagnosis, treatment, and research, accounting for up to 25% of global cancer cases. Due to their rarity and atypical presentations, they are often misdiagnosed, resulting in late-stage detection and poor outcomes. Here, we describe a patient case with advanced metastatic nasopharynx NUT carcinoma, one of the rarest and most aggressive cancers. We conducted a comprehensive analysis of this patient’s tumor, including Tumor Mutation Burden, Microsatellite Instability, and genetic profiling to explore further putative druggable targets. The tumor exhibited high PD-L1 expression and showed a notable response to immune checkpoint inhibitors when combined with platinum-based radio-chemotherapy. Our findings indicate that checkpoint inhibitors could play a critical role in treating NUT carcinoma, offering new therapeutic avenues and hope for patients with this challenging diagnosis. Whether PD-L1 expression may be a useful predictor of immune checkpoint inhibitor efficacy warrants further research.

NUT carcinoma (NC) is a highly aggressive malignancy characterized by an oncogenic fusion gene incorporating the NUTM1 gene. To date, no established treatment options exist. CRISPR-Cas9 technology allows precise genomic targeting, thereby presenting a promising therapeutic strategy for cancers with well-defined genomic alterations such as oncogenic fusion genes. In this study, we investigated the effects of CRISPR-Cas9-mediated disruption of the BRD4::NUTM1 fusion gene in NC cell lines using multiple single guide RNAs (sgRNAs) to target different sites of both fusion partner genes. Our experiments identified promising sgRNA candidates that were shown to successfully disrupt the BRD4::NUTM1 fusion gene at the DNA level, thus leading to an efficient knockout of the aberrant fusion protein. This genetic disruption resulted in profound functional impairments in NC cells, which included a significant reduction in proliferative capacity, cell-cycle arrest, and induction of apoptosis. These findings underscore the dependency of NC on the BRD4::NUTM1 fusion gene and highlight the potential of CRISPR-based strategies for targeting cancer at its genetic level. This approach also holds promise for the development of highly specific and effective therapies for many other oncogenic fusion driven cancers.

Background and Aims: NUT carcinoma (NC) is a sporadic, highly aggressive tumor that primarily affects children, adolescents, and young adults and is characterized by the presence of somatic NUTM1 rearrangements. This analysis by the European Cooperative Study Group for Pediatric Rare Tumors (EXPeRT) aims to fill the knowledge gap regarding the clinical characteristics of children with NC. Methods: A retrospective case series of NC-patients aged 0-18 years treated between 2011 and 2023 was conducted using the EXPeRT database. Relevant clinical characteristics, including treatment and outcome were recorded. Results: Twenty-seven patients with a median age of 13 years (range 7-18) were analyzed. Thirteen patients were initially misdiagnosed. Sixteen patients had thoracic and 11 extra-thoracic tumors, including three in the nasal/ sinus region and two in the submandibular glands. Despite intense multimodal treatment, median event-free and overall survivals were 1.5 and 6.5 months, respectively. Conclusions: Early diagnosis of NC by examination of the NUTM1 rearrangement in undifferentiated or poorly differentiated carcinomas is crucial in order to initiate specific and intensive therapy as quickly as possible. Similar to adult patients, only a minority of pediatric patients achieved prolonged survival. Therefore, the development of novel therapeutic strategies in future joint clinical trials is essential.

Aim/Introduction: Peptide receptor radionuclide therapy (PRRT) represents a cornerstone of treatment regimens for patients with low proliferative neuroendocrine tumors (NETs). However, in patients experiencing somatostatin receptor-positive NET with higher proliferation rates, a value and potential therapeutic benefit of PRRT as part of multimodal treatment approaches and potentially with addition of radiosensitizing agents has not yet been established. Patients and Methods: In this study, 20 patients with histologically confirmed gastroenteropancreatic (GEP) NET with proliferation rates (Ki67) between 15% and 55% were treated either with PRRT only (n = 10) or with a combination therapy (n = 10) comprising PRRT and capecitabine/temozolomide (CAP/TEM) for at least 2 consecutive cycles. Results: Disease control rate in patients treated with PRRT alone was 60% (40% stable disease and 20% partial response). Strikingly, in patients treated with PRRT in combination with radiosensitization (CAP/TEM), the disease control rate was 90% (20% stable disease and 70% partial response). The median progression-free survival in the PRRT only group was 12 months, whereas the median progression-free survival in the PRRT + CAP/TEM group was 26 months and has not been yet reached for all patients in the group during the observation period. The median disease-specific survival for patients with PRRT alone was 51 months, whereas this end point was not yet reached in the PRRT + CAP/TEM group. Moreover, the PRRT + CAP/TEM group showed a significantly higher reduction of SSTR-PET-based metabolic tumor volume and chromogranin A levels compared with the PRRT only group. Importantly, adverse events of all grades did not differ between both groups. Conclusions: PRRT + CAP/TEM represents a highly promising and well-tolerated therapeutic regimen for patients experiencing somatostatin receptor-positive NET with higher (Ki67 >= 15%) proliferation rate. Prospective randomized clinical trials are warranted. [GRAPHICS] .

NUT carcinomas (NC) are very rare and highly aggressive tumors, molecularly defined by an aberrant gene fusion involving the NUTM1 gene. NCs preferentially arise intrathoracically or in the head and neck region, having a highly adverse prognosis with almost no long-term survivors. Here, we report on a cohort of 35 adult NC patients who were evaluated at University Hospital Tuebingen in an eight year time span, i.e. between 2016 and 2023. Primary objectives were overall survival (OS) and influence of primary tumor locations, fusion gene types and staging on OS. Secondary objectives were patient baseline characteristics, risk factors, tumor markers, treatment decisions and responses to therapy comparing thoracic vs non -thoracic origins. Further, data from tumor genome sequencing were analyzed. In this monocentric German cohort, 54 % of patients had thoracic tumors and 65 % harbored a BRD4-NUTM1 fusion gene. Median OS was 7.5 months, being significantly dependent on primary tumor location and nodal status. Initial misdiagnosis was a problem in 31 % of the cases. Surgery was the first treatment in most patients (46 %) and 80 % were treated with polychemotherapies, showing longer progression free survival (PFS) with ifosfamide-based than with platinum -based regimens. Patients treated with an immune checkpoint inhibitor (ICI) in addition to first -line chemotherapy tended to have longer OS. Initial LDH levels could be identified as a prognostic measure for survival prognosis. Sequencing data highlight aberrant NUTM1 fusion genes as unique tumor driver genes. This is the largest adult European cohort of this orphan tumor disease, showing epidemiologic and molecular features as well as relevant clinical data. Awareness to prevent misdiagnosis, fast contact to a specialized nationwide center and referral to clinical studies are essential as long-term survival is rarely achieved with any of the current therapeutic regimes.

Ewing sarcoma is the second most common primary malignant bone cancer in children and adolescents. This rare type of cancer is characterized by its high malignancy and therefore high risk of metastases. Typically, Ewing sarcomas originate from bones. However, extraosseous Ewing sarcoma such as pulmonary Ewing sarcoma can also be found. In this case report, we present a 55-year old male patient who was initially diagnosed with non-small cell lung cancer at his local district hospital. However, the diagnosis was changed to one of pulmonary Ewing sarcoma after subsequent histopathological and molecular pathological analysis performed in a reference pathology laboratory. After patient referral to a certified (according to the German Cancer Society) high-volume sarcoma center, multimodal chemotherapy was initiated based on recently published clinical data as opposed to the more commonly used treatment regimen in Europe. The patient responded well to treatment and underwent a complete surgical tumor resection followed by radiotherapy. In summary, this case report highlights the importance of a rigorous and timely histopathological examination of biopsy samples by a specialized cancer center to enable a correct diagnosis of the cancer type. Additionally, molecular pathology plays a crucial part in this analysis and allows the necessary differentiation between cancer types. Up to now, there is no international treatment guideline available for the treatment of Ewing sarcoma. Patients should be referred to specialist centers to allow the best possible treatment of the cancer type in view of current published clinical data. In the case of Ewing sarcoma, and in accordance with the most recent research, patients should be treated with vincristine, doxorubicin and cyclophosphamide plus ifosfamide and etoposide in combination with local treatment such as surgery and/or radiotherapy because this has been demonstrated to be the more effective therapy.

Mutations of β-catenin are frequent in HCC and are involved in dysfunctional antigen presentation, immunosuppression, and insensitivity to immunotherapies. Animal models of HCC with defined immunogenicity and dysfunctional Wnt/β-catenin activation are lacking that are suitable for investigations on local immunotherapies including virotherapy. First, we induced HCC In C57BL/6 mice in-situ by hydrodynamic injection (HDI) or local electroporation (EP) of plasmids to deliver SB13-transposase and transposons encoding c-Myc, shRNAp53, mutated p53-R172H, with or without ΔN90-β-catenin. To introduce immunogenicity, c-myc expression was genetically linked to immunogenic (neo)epitope arrays. After HDI, ΔN90-β-cat expression was essential for develoment of immunogenic HCCs. After EP, HCC occurred in both groups but significantly faster and more reliable in the presence of ΔN90-β-cat. Reduction of EP-induced tissue damage by lowering the voltage almost fully prevented tumor development without mutant β-cat suggesting that the presence of mutated β-cat was decisive in this setting to enable tumor escape from immune surveillance. For a s.c. tumor model, we established the primary cell line HepM-683 from ΔN90-β-cat activated HCC. In this model, we investigated local virotherapy using the oncolytic adenovirus hTert-Ad together with various adenoviral vectors for immunoactivating cytokines. Compared with virotherapy alone, additional expression of Mip1α, Flt3L together with Xcl1, or CCL5 respectively, showed successful TME activation and resulted in reduced growth of β-cat-dysfunctional tumors. Together, we have established syngeneic in-situ and s.c. murine HCC models featuring immunogenic epitopes and β-cat-dependent growth characteristics. These models are suitable for investigations of local immunotherapies such as virotherapy to overcome mut-β-cat-dependent immunosuppression in HCC.

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.

Several studies have demonstrated, both in vitro and in animal models, the anti-tumor efficacy of high-dose ascorbate treatment against a variety of tumor entities, including glioblastoma, the most common and aggressive primary malignant brain tumor. The aim of this study was to investigate the effects of high-dose ascorbate as well as dehydroascorbic acid on human glioblastoma cell lines and to evaluate different treatment conditions for the combined administration of ascorbate with magnesium (Mg2+) and iron (Fe3+). Intracellular levels of reactive oxygen species and the induction of cell death following ascorbate treatment were also investigated. We demonstrated high cytotoxicity and antiproliferative efficacy of high-dose ascorbate in human glioblastoma cells, whereas much weaker effects were observed for dehydroascorbic acid. Ascorbate-induced cell death was independent of apoptosis. Both the reduction in cell viability and the ascorbate-induced generation of intracellular reactive oxygen species could be significantly increased by incubating the cells with Fe3+ before ascorbate treatment. This work demonstrates, for the first time, an increase in ascorbate-induced intracellular ROS formation and cytotoxicity in human glioblastoma cells by pre-treatment of the tumor cells with ferric iron, as well as caspase-3 independence of cell death induced by high-dose ascorbate. Instead, the cell death mechanism caused by high-dose ascorbate in glioblastoma cells shows evidence of ferroptosis. The results of the present work provide insights into the efficacy and mode of action of pharmacological ascorbate for the therapy of glioblastoma, as well as indications for possible approaches to increase the effectiveness of ascorbate treatment.

IntroductionNeuroendocrine neoplasms (NEN) are a rare and heterogenous group of tumors arising from neuroendocrine cells in multiple organs. Neuroendocrine tumors (NET) G3 encompass a small subgroup accounting for less than 10% of all neuroendocrine neoplasms. In contrast to NET G1 and G2 as well as neuroendocrine carcinomas (NEC), in NET G3 data on treatment and patient outcomes are still limited. Especially in a metastasized tumor stage, the role of surgery, peptide receptor radionucleotide therapy (PRRT), and systemic chemotherapy is not clearly defined.MethodsIn this real-life cohort, we consecutively analyzed clinical outcome in NET G3 patients receiving different diagnostic and treatment.Results and discussionWe found that even metastasized NET G3 patients undergoing surgery, or receiving radiation, somatostatin analogues (SSA), and PRRT showed a clear survival benefit. Interestingly, all treatment regimen were superior to classical chemotherapeutic agents. In addition, somatostatin receptor (SSTR) PET-CT, FDG PET-CT, and repetitive biopsies were shown to be useful diagnostic and prognostic tools in NET G3. Our study demonstrates that patients with highly proliferative NET G3 might benefit from less aggressive treatment modalities commonly used in low proliferative NEN.

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.

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.