Therapeutic activity of retroviral replicating vector-mediated gene therapy in combination with anti-PD-1 antibody in a murine pancreatic cancer model.

Toca 511, a tumor-selective retroviral replicating vector encoding the yeast cytosine deaminase (yCD) gene, exerts direct antitumor effects through intratumoral prodrug 5-fluorocytosine (5-FC) conversion to active drug 5-fluorouracil by yCD, and has demonstrated therapeutic efficacy in preclinical and clinical trials of various cancers. Toca 511/5-FC treatment may also induce antitumor immunity. Here, we first examined antitumor immune responses activated by Toca 511/5-FC treatment in an immunocompetent murine pancreatic cancer model. We then evaluated the therapeutic effects achieved in combination with anti-programmed cell death protein 1 antibody. In the bilateral subcutaneous tumor model, as compared with the control group, enhanced CD8+ T-cell-mediated cytotoxicity and increased T-cell infiltration in Toca 511-untransduced contralateral tumors were observed. Furthermore, the expression levels of T-cell co-inhibitory receptors on CD8+ T-cells increased during treatment. In the bilateral subcutaneous tumor model, combination therapy showed significantly stronger tumor growth inhibition than that achieved with either monotherapy. In an orthotopic tumor and peritoneal dissemination model, the combination therapy resulted in complete regression in both transduced orthotopic tumors and untransduced peritoneal dissemination. Thus, Toca 511/5-FC treatment induced a systemic antitumor immune response, and the combination therapy could be a promising clinical strategy for treating metastatic pancreatic cancer.

Mixed invasive mucinous and non-mucinous adenocarcinoma of the lung with hematogenous metastases to multiple organs.

Mixed invasive mucinous and non-mucinous adenocarcinoma is a rare variant of lung adenocarcinoma. In pure invasive mucinous adenocarcinoma, multilobar and bilateral involvement are common, and extrathoracic metastasis is rare. Here, we report a case of mixed invasive mucinous and non-mucinous adenocarcinoma with distant metastasis to multiple organs without marked enlargement of the primary lung lesion. The pathological findings indicated high tumor invasiveness and the patient died 10 months after diagnosis despite chemoimmunotherapy. Further investigations are necessary to elucidate the clinical characteristics and appropriate management of mixed invasive mucinous and non-mucinous adenocarcinoma.

Invasive thymoma metastases to the pancreas: A case report.

INTRODUCTION AND IMPORTANCE: Thymoma is the most common type of tumor that develops in the thymic epithelial cells. Although thymomas can invade surrounding organs in the chest cavity, extrathoracic metastasis is very rare, and little is known about the prognosis and effective treatments for such tumors. Herein, we report a case of an invasive thymoma metastasizing to the pancreas after incomplete resection. CASE PRESENTATION: A 47-year-old man presented to our hospital with an anterior mediastinal mass. Although a thymic tumor was suspected, complete resection was not achieved because the tumor had invaded the pulmonary artery trunk, superior pulmonary vein, and left brachiocephalic vein. The pathological diagnosis was a Type B3 thymoma. The patient underwent chemotherapy and radiotherapy after surgery. Three years after surgery, computed tomography indicated a pancreatic mass suggestive of pancreatic cancer. Distal pancreatectomy was performed after neoadjuvant chemotherapy and radiotherapy. The pancreatic mass was diagnosed as Type B3 thymoma metastasis. Thirteen months after surgery for the pancreatic lesion, the patient underwent resection of the bilateral lung metastases. CLINICAL DISCUSSION: To the best of our knowledge, only four cases of metastatic thymic tumors in the pancreas have been reported. All patients who underwent surgical resection for pancreatic metastasis survived for >6 months including our case. CONCLUSION: In cases of thymic tumors with metastasis to extra-thoracic organs, complete resection of the metastatic lesions can contribute to prolonged survival.

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

Myasthenia Gravis Associated With Pembrolizumab for Relapsed Lung Cancer After Thymoma Resection.

Immunotherapy has demonstrated clinical efficacy in patients with thymic epithelial tumors; however, there is the potential risk of serious immune-related adverse events (irAEs). Here, we report a case of myasthenia gravis (MG) associated with pembrolizumab treatment that developed after thymoma resection in a patient with lung adenocarcinoma. Symptoms of MG occurred 16 days after pembrolizumab administration and progressed rapidly, necessitating mechanical ventilation and tracheostomy. Even after tumor resection, careful monitoring is crucial for patients with thymic tumors being managed with immune checkpoint therapy, particularly regarding the development of severe irAEs.

Retroviral Replicating Vector Toca 511 (Vocimagene Amiretrorepvec) for Prodrug Activator Gene Therapy of Lung Cancer.

Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.

Small-sized peripheral squamous cell lung carcinoma with chest wall invasion.

In lung cancer, chest wall infiltration caused by a tumor with a small diameter is extremely rare. The pathophysiologic features and prognosis of this phenomenon are poorly understood. Here, we report on a case in which a small peripheral lung cancer showed marked invasion into the chest wall. Although complete resection and postoperative adjuvant treatment were performed, lymph node recurrence developed and the patient died in one and a half years. Peripheral lung cancer can show exophytic development and infiltration of the chest wall, leading to poor prognosis, even if the tumor size is relatively small.

Antibody responses induced by the BNT162b2 mRNA COVID-19 vaccine in healthcare workers in a single community hospital in Japan.

INTRODUCTION: The effectiveness of several vaccines against coronavirus disease (COVID-19) has been reported in the real-world setting. However, it is still unknown how long antibodies persist following vaccination and whether or not the persistence of antibodies has a protective effect against COVID-19. METHODS: Healthcare workers who had received two doses of the BNT162b2 mRNA COVID-19 vaccine were enrolled, and a single-center study was conducted at the National Hospital Organization Hakodate National Hospital. Serum samples from all participants were collected 13-21 weeks (median: 20 weeks) after the second dose of vaccination. The antibody titers were measured using an electrochemiluminescence immunoassay (Elecsys® Anti-SARS-CoV-2 S). Data on characteristics of the participants were gathered from patient records and interview sheets. RESULTS: A total of 401 participants, among whom 70.1% were women and the median age was 42 years, were evaluated in this study. None of the participants had a definite COVID-19 history, and all participants who received complete vaccination showed positive antibody titers. The antibody titer was observed to be higher in participants with younger age (p < 0.001) and those who were females (p = 0.028). Despite the higher risk of infection than that of the general public, no vaccinated staff developed breakthrough infections. CONCLUSIONS: This study demonstrates the significant contribution of the BNT162b2 vaccine in the acquisition of anti-SARS-CoV-2S antibodies; therefore, the general population should benefit from these two vaccine doses, which are expected to be protective for at least five months.

Primary undifferentiated pleomorphic sarcoma (Malignant fibrous histiocytoma) of the lung: A case report.

Primary UPS of the lung, so-called MFH, is a rare aggressive neoplasm and known to be a high risk of recurrence and metastasis. Surgical resection without residual tumor is the main option of treatment and the best chance for long-term survival.

Therapeutic activity of retroviral replicating vector-mediated prodrug activator gene therapy for pancreatic cancer.

Toca 511, a retroviral replicating vector (RRV) encoding the yeast cytosine deaminase (yCD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU), is currently being evaluated in Phase II/III clinical trials for glioma, and showing highly promising evidence of therapeutic activity. Here we evaluated RRV-mediated prodrug activator gene therapy as a new therapeutic approach for pancreatic ductal adenocarcinoma (PDAC). RRV spread rapidly and conferred significant cytotoxicity with prodrug in a panel of PDAC cells. Efficient intratumoral replication and complete inhibition of tumor growth upon 5-FC administration were observed in both immunodeficient and immunocompetent subcutaneous PDAC models. Biodistribution of RRV was highly restricted in normal tissues, especially in immunocompetent hosts. Tumor growth inhibition by Toca 511 followed by 5-FC was also confirmed in the orthotopic PDAC model. This study provides the first proof-of-concept for application of Toca 511 and Toca FC (extended release 5-FC) to the treatment of human PDAC, and provided support for inclusion of PDAC in a Phase I study evaluating Toca 511 in various systemic malignancies, (NCT02576665), which has recently been initiated.

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity.

BACKGROUND: Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects. METHODS: Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts. RESULTS: In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1-2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells. CONCLUSION: These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model.

BACKGROUND: Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511-expressing gliomas in a syngeneic mouse model. METHODS: Flow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC. RESULTS: Tumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma-expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell-dependent protection from future tumor challenge. CONCLUSIONS: Treatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

CD40 Expression in Human Esophageal Squamous Cell Carcinoma Is Associated with Tumor Progression and Lymph Node Metastasis.

BACKGROUND: The co-stimulatory molecule cluster of differentiation 40 (CD40) is widely expressed in various types of malignant tumors, but its role remains unclear. The purpose of this study was to investigate the relationship between CD40 expression and clinicopathological variables in patients with esophageal squamous cell carcinoma (ESCC), as well as the function of CD40 expressed on ESCC tumor cells in vitro. MATERIALS AND METHODS: Tumor specimens of patients who underwent surgical resection for ESCC were immunohistochemically analyzed for CD40 expression. RESULTS: Of the 122 specimens, 45 (37%) were positive for CD40. Significant positive correlation was found between CD40 expression and p-stage (p=0.0011), histopathological grade (p=0.0143), pT-classification (p=0.0011), and pN-classification (p=0.0007). Survival of patients with stage III and IV disease with positive CD40 expression was significantly shorter than that of those with negative expression (log-rank test, p=0.0422). In in vitro analysis, while the addition of recombinant human CD154 did not inhibit growth, it did induce a significant increase in interleukin 6 production in ESCC cell lines. CONCLUSION: These results suggest that functional expression of CD40 on tumor cells might play an important role in tumor progression and lymph node metastasis in ESCC.

Inhibition of Eph receptor A4 by 2,5-dimethylpyrrolyl benzoic acid suppresses human pancreatic cancer growing orthotopically in nude mice.

Ephrin receptor A4 (EphA4) is overexpressed in human pancreatic adenocarcinoma (PDAC) and activate cell growth. Recent studies have identified small molecules that block EphA4. In this study, we investigated the correlation between EphA4 expression and the prognosis of patients with PDAC. We also examined the cytostatic efficacy of 2,5-dimethylpyrrolyl benzoic acid (compound 1), a small molecule that blocks EphA4, in PDAC cells. Overall survival of patients with EphA4 positivity was significantly shorter than that of patients with EphA4 negativity (P = 0.029). In addition, multivariate analysis revealed that EphA4 expression was an independent prognostic factor in PDAC patients (P = 0.039). Compound 1 showed a cytostatic efficacy in PDAC cells expressing EphA4 in vitro and in vivo. Our study indicated that compound 1 suppressed both EphA4 and Akt phosphorylations, and induced apoptosis in PDAC cells expressing EphA4. In conclusion, compound 1 has a high potential as a therapeutic agent for patients with PDAC.

Bronchogenic cyst associated with congenital absence of the pericardium.

We report a rare case of bronchogenic cyst associated with congenital complete absence of the pericardium. A 17-year-old male was admitted to the hospital for surgical resection of a growing cystic lesion located in the middle mediastinum. The patient was asymptomatic and no significant findings were found on physical examination. Resection of the mediastinal cyst was performed by video-assisted thoracoscopic surgery. The complete absence of the pericardium was immediately observed along with a cystic tumor arising from the mediastinum. After the resection of the cyst, no additional procedure to reconstruct the absence of the left pericardium was performed. Pathological diagnosis was a bronchogenic cyst. Congenital absence of the pericardium may be associated with the bronchogenic cyst and complete absence of the pericardium requires no additional surgical reconstruction, if remaining space in the pleural cavity is small enough to avoid cardiac disposition after surgical resection of the cyst.

Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.

Mucosal gene therapy using a pseudotyped lentivirus vector encoding murine interleukin-10 (mIL-10) suppresses the development and relapse of experimental murine colitis.

BACKGROUND: Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis. METHODS: Lentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-α and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR. RESULTS: Mucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-α and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected. CONCLUSIONS: Topically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis.

Modified Robicsek procedure for pectus excavatum in adult patients.

OBJECTIVES: The aim of this study was to clarify the short-term and mid-term outcomes of the modified Robicsek procedure using polypropylene mesh for adult pectus excavatum patients. METHODS: Retrospective chart review was performed. Between 2001 and 2012, 46 consecutive adult patients underwent modified Ravitch repair using polypropylene mesh for pectus excavatum at our institution. There were 30 males and 16 females, with a median age of 25.5 (range: from 17 to 60). Potential risk factors for perioperative complications and early failure in pectus repair were analysed with χ(2) test or Fisher's exact test and Mann-Whitney test. Potential risk factors for postoperative mid-term recurrence were analysed using Cox proportional hazard regression model. RESULTS: The postoperative follow-up interval was 7.0 ± 15.1 months (mean ± standard deviation). No mortality and 11 patients (24.0%) of morbidity, including 2 patients with temporary mesh infection, were noted. A lower preoperative %FEV1.0 predicted was a significant factor (P = 0.0088) of morbidity in multivariate analysis. Regarding chest morphology, early failure in 1 patient (2.2%) and mid-term recurrence in 1 patient (2.2%) were seen. Previous pectus repair (P = 0.0324) and severe asymmetry (P = 0.04) were significant factors of early failure in multivariate analysis, while no significant factor for mid-term recurrence was found. CONCLUSIONS: Modified Robicsek procedure with a polypropylene mesh is associated with low incidences of early failure and mid-term recurrence. The procedure is recommended for adult pectus excavatum patients, including those with asymmetric or combined deformity as an initial or reoperative procedure.

Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function.

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence.