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After the discovery and characterization of the adenovirus in the 1950s, this prevalent cause of the common cold and other usually mild diseases has been modified and utilized in biomedicine in several ways. To date, adenoviruses are the most frequently used vectors and therapeutic (e.g., oncolytic) agents with a number of beneficial features. They infect both dividing and nondividing cells, enable high-level, transient protein expression, and are easy to amplify to high concentrations. As an important and versatile research tool, it is of essence to understand the limits and advantages that genetic modification of adenovirus vectors may entail. Therefore, a retrospective analysis was performed of adenoviral gene therapy constructs produced in the same laboratory with similar methods. The aim was to assess the impact of various modifications on the physical and functional titer of the virus. It was found that genome size (designed within "the 105% golden rule") did not significantly affect the physical titer of the adenovirus preparations, regardless of the type of transgene (e.g., immunostimulatory vs. other), number of engineered changes, and size of the mutated virus genome. One statistically significant exception was noted, however. Chimeric adenoviruses (5/3) had a slightly lower physical titer compared to Ad5-based viruses, although a trend for the opposite was true for functional titers. Thus, 5/3 chimeric viruses may in fact be appealing from a safety versus efficacy viewpoint. Armed viruses had lower functional and physical titers than unarmed viruses, while five genomic modifications started to decrease functional titer. Importantly, even highly modified armed viruses generally had good titers compatible with clinical testing. In summary, this paper shows the plasticity of adenovirus for various vector, oncolytic, and armed oncolytic uses. These results inform future generations of adenovirus-based drugs for human use. This information is directly transferable to academic laboratories and the biomedical industry involved in vector design and production optimization.

The development of treatments for attention impairments is hampered by limited knowledge about the malleability of underlying neural functions. We conducted the first randomized controlled trial to determine the modulations of brain activity associated with working memory (WM) training in adults with attention‐deficit hyperactivity disorder (ADHD). At baseline, we assessed the aberrant functional brain activity in the n‐back WM task by comparing 44 adults with ADHD with 18 healthy controls using fMRI. Participants with ADHD were then randomized to train on an adaptive dual n‐back task or an active control task. We tested whether WM training elicits redistribution of brain activity as observed in healthy controls, and whether it might further restore aberrant activity related to ADHD. As expected, activity in areas of the default‐mode (DMN), salience (SN), sensory‐motor (SMN), frontoparietal (FPN), and subcortical (SCN) networks was decreased in participants with ADHD at pretest as compared with healthy controls, especially when the cognitive load was high. WM training modulated widespread FPN and SN areas, restoring some of the aberrant activity. Training effects were mainly observed as decreased brain activity during the trained task and increased activity during the untrained task, suggesting different neural mechanisms for trained and transfer tasks., We conducted the first randomized controlled trial to determine the modulations of brain activity associated with working memory (WM) training in adults with attention‐deficit hyperactivity disorder. WM training modulated widespread areas in the frontoparietal and salience networks, restoring some of the aberrant activity. Training effects were mainly observed as decreased brain activity during the trained task and increased activity during the untrained task, suggesting different neural mechanisms for trained and transfer tasks.

BACKGROUND CONTEXT: The leading surgical treatment of cervical radiculopathy is anterior cervical discectomy and fusion (ACDF). However, it has been suggested that ACDF procedures could lead to accelerated degeneration of the adjacent cervical discs (adjacent segment disease, or ASD) and the effect of ACDF surgery on neck symptoms and quality of life in the long term is not fully understood. Patients operated on at young ages generally have a long life expectancy and a long number of working years ahead of them. Thus, this patient group is of special interest when considering the accumulation of cervical problems due to possible ASD, the overall progressive nature of cervical degeneration in the long term, and their effects on related quality of life. PURPOSE: Our goal was to study the health-related quality of life in the long-term follow-up after ACDF surgery in the young adult population between the ages of 18 and 40. STUDY DESIGN: A retrospective cohort study with propensity matched controls. PATIENT SAMPLE: All patients between 18 and 40 years of age at the time of the surgery who underwent ACDF due to degenerative cervical disease at Helsinki University Hospital between the years 1990 and 2005 who had filled in the quality of life questionnaires 12 to 28 years after the surgery (281 patients), and a propensity matched control cohort of the general population selected based on age, sex, and smoking status. OUTCOME MEASURES: Quality of life measured by the EuroQol questionnaire (EQ-5D-3L and EQ-VAS). METHODS: The medical records of all patients who underwent ACDF due to degenerative cervical disease at the age of 18 to 40 years at Helsinki University Hospital between 1990 and 2005 were analyzed retrospectively. The EuroQol questionnaire was sent to all patients whose contact information could be obtained (443 patients) at the end of the follow-up (median 17.5 years) to assess their current quality of life. A total of 281 patients returned the questionnaires and were included in this study. Quality of life was compared to that in the general Finnish population using a similar sized control cohort selected through propensity matching. RESULTS: The patients who had undergone ACDF surgery reported significantly more problems than the general population cohort in three out of five dimensions that were assessed in the EQ-5D questionnaire, including mobility, usual activities, and pain/discomfort. Similarly, the overall EQ-5D-3L index calculated from the dimensional values was lower (0.74 vs. 0.83, p=.000), depicting a generally decreased health-related quality of life among patients. Spondylosis as a primary diagnosis, clinical myelopathy, and further cervical surgeries were associated with lower quality of life in the subgroup analyses of the patients. Similarly, in the EQ-VAS assessment, patient subgroups with spondylosis as a primary diagnosis, at least one reoperation, operation on more than one level, and clinical myelopathy were associated with lower scores and lower quality of life. The mean EQ-VAS score among patients was 73%. Regardless of the decreased health-related quality of life, there was no statistically significant difference in the concurrent employment status between the patient and control groups. CONCLUSIONS: The health-related life quality measured by the EQ-5D-3L was lower in the patient population than in the general population. Patients had more problems with mobility and usual activities and more pain/discomfort. However, satisfaction with the surgery was very high, and there was no significant difference in employment status between the patients and the control population. Patients with spondylosis as a primary diagnosis had lower quality of life compared to patients with disc herniation. Also, clinical myelopathy and further cervical surgeries during follow-up were associated with lower quality of life in the subgroup analyses of the patients. It must also be kept in mind that we do not know what the situation could have been without surgery and with conservative treatment only. (C) 2021 The Authors. Published by Elsevier Inc.

Anterior cervical discectomy and fusion (ACDF) procedures is thought to lead to accelerated degeneration of the adjacent cervical discs and in some cases can be symptomatic (adjacent segment disease, or ASD). The occurrence of ASD is of particular interest when treating young individuals, as the cumulative disease burden may become increasingly significant during their expectedly long lifetime. However, the overall impact of a surgical intervention on the lifetime prognosis of ASD remains unclear.Our goal was to study the long-term outcomes of ACDF surgery among those members of the young adult population who have been operated on between the ages of 18 and 40.Retrospective study.All patients between 18 and 40 years of age at the time of surgery who underwent ACDF due to degenerative cervical disorders at Helsinki University Hospital between the years of 1990 and 2005 (476 patients).Cervical reoperation rate, satisfaction with the surgery, employment status, Neck Disability Index (NDI).We retrospectively analyzed the medical records of all patients between 18 and 40 years of age at the time of surgery who underwent ACDF due to degenerative cervical disorders at Helsinki University Hospital between the years of 1990 and 2005. We sent questionnaires to all available patients at the end of the follow-up (median 17.5 years) to assess their current neck symptoms, general situations, and levels of satisfaction with the surgery. Furthermore, we compared the results for different types of ACDF surgeries (ie, discectomy only vs. synthetic cage or bone autograft implantation for fusion) in propensity-score-matched groups.Of the 476 patients who were included in the study, surgery was performed in 72% of the cases due to intervertebral disc herniation and in 28% due to spondylotic changes. The total reoperation rate during the entire follow-up (median 17.5 years) was 24%, and 19.5% if early reoperations (28 days from index surgery) were excluded. At 10 years postsurgery, the total reoperation rate was 16.8% and 12.8% with early reoperations excluded. The probability of surgery for adjacent level disease was 10.3% at 10 years and 16.8% for the duration of the entire follow-up, with the annual incidence rate of 1.1% for those with ASD requiring surgery. Statistically significant risk factors leading to the need for further cervical surgery included central spinal cord compression and smoking at the time of the index operation. After propensity score matching, there was no significant difference found between the outcomes of different types of surgery. A total of 443 patients were still able to be contacted 12-28 years after the surgery. Of the 281 patients responding to the questionnaires, 92% were still satisfied with the results. With respect to employment, 67% of patients were working, 7% were unemployed, and 7% were on disability due to cervical problems. The median NDI score was 12%, with 56% of patients having an NDI score lower than 15%; it has been suggested that this latter NDI score serves as a cut-off value for significant neck morbidity. The NDI scores were significantly higher among female patients, patients with spondylosis, and patients having undergone further cervical surgeries during the follow-up.Long-term satisfaction with the surgery was very high, and the employment rate among patients resembled that of the general population in Finland. Thus, the long-term prognosis after having ACDF surgery at a younger age seems to be good, even though nearly half of the patients experienced some persistent neck symptoms later in life.

The first US Food and Drug Administration (FDA)- and EMA-approved oncolytic virus has been available since 2015. However, there are no markers available that would predict benefit for the individual patient. During 2007–2012, we treated 290 patients with advanced chemotherapy-refractory cancers, using 10 different oncolytic adenoviruses. Treatments were given in a Finnish Medicines Agency (FIMEA)-regulated individualized patient treatment program (the Advanced Therapy Access Program [ATAP]), which required long-term follow-up of patients, which is presented here. Focusing on the longest surviving patients, some key clinical and biological features are presented as “oncograms.” Some key attributes that could be captured in the oncogram are suggested to predict treatment response and survival after oncolytic adenovirus treatment. The oncogram includes immunological laboratory parameters assessed in peripheral blood (leukocytes, neutrophil-to-lymphocyte ratio, interleukin-8 [IL-8], HMGB1, anti-viral neutralizing antibody status), features of the patient (gender, performance status), tumor features (histological tumor type, tumor load, region of metastases), and oncolytic virus-specific features (arming of the virus). The retrospective approach used here facilitates verification in a prospective controlled trial setting. To our knowledge, the oncogram is the first holistic attempt to identify the patients most likely to benefit from adenoviral oncolytic virotherapy. Keywords: oncolytic adenovirus, oncolytic virotherapy, immunotherapy, cancer, immunostimulation, anti-cancer, adenovirus, oncoimmunology, immunogram, oncogram

Hydrocephalic patients with abdominal pathologies often need a ventriculo-atrial (VA) shunt placement. Cut-down on the internal jugular vein has historically been used to insert a VA shunt. This technique is more time consuming and has greater complications. Less invasive methods, such as ultrasound-guided percutaneous VA shunt placement provides greater comfort for surgeon, is more rapid, and has fewer complications. However, this technique has not been demonstrated on video. Here we demonstrate ultrasound-guided and ECG-aided VA shunt catheter placement in a 70-year-old patient with normal pressure hydrocephalus. The internal jugular vein is punctured under ultrasound guidance with an 18-gauge needle. A guidewire is introduced through the needle, the needle is removed, and a small skin incision is placed at the entry point of the guidewire. A skin dilator with a sheath introducer is advanced to the vein using the guidewire and the guidewire is thereafter removed. An atrial shunt catheter (e.g. Codman® Medos® Atrial catheter) filled with sterile water is inserted through the sheath. The sheath is removed and a syringe filled with sterile aqua is connected to the catheter with a metal tip. The ECG connection of the right upper limb is connected to the tip of syringe to adjust for the optimal depth of the catheter under ECG guidance (point of highest p-wave amplitude). The catheter is clamped and tunneled to reach the site for the valve on the scalp. The ventricle catheter is placed at the Kocher point and connected to the valve (Video 1). Conclusion: Ultrasound-guided VA shunt placement is safe, comfortable, rapid, and has a reduced rate of complications. Keywords: Ventriculo-atrial shunt placement, Surgery, Technical aspects, Video demonstration

In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients.

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.

// Otto Hemminki 1 , Suvi Parviainen 1 , Juuso Juhila 1 , Riku Turkki 2 , Nina Linder 2 , Johan Lundin 2, 3 , Matti Kankainen 4 , Ari Ristimaki 5 , Anniina Koski 1 , Ilkka Liikanen 1 , Minna Oksanen 1 , Dirk M. Nettelbeck 6 , Kalevi Kairemo 7 , Kaarina Partanen 7 , Timo Joensuu 7 , Anna Kanerva 1, 8 , Akseli Hemminki 1, 7, 9 1 Cancer Gene Therapy Group, Transplantation Laboratory & Haartman Institute, University of Helsinki, Helsinki, Finland 2 Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland 3 Division of Global Health/IHCAR, Karolinska Institutet, Stockholm, Sweden 4 CSC - IT Center for Science Ltd, Helsinki, Finland 5 Department of Pathology, HUSLAB and Haartman Institute, Helsinki, University Central Hospital and Genome-Scale Biology, Research Programs Unit, University of Helsinki, Helsinki, Finland 6 German Cancer Research Center (DKFZ), Heidelberg, Germany 7 Docrates Cancer Center, Helsinki, Finland 8 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Helsinki, Finland 9 TILT Biotherapeutics Ltd, Helsinki, Finland Correspondence to: Akseli Hemminki, e-mail: akseli.hemminki@helsinki.fi Keywords: Oncolytic, immunotherapy, cancer, ATAP Received: September 17, 2014 Accepted: December 14, 2014 Published: February 24, 2015 ABSTRACT Oncolytic viruses that selectively replicate in tumor cells can be used for treatment of cancer. Accumulating data suggests that virus induced oncolysis can enhance anti-tumor immunity and break immune tolerance. To capitalize on the immunogenic nature of oncolysis, we generated a quadruple modified oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GMCSF). Ad5/3-E2F-Δ24-GMCSF (CGTG-602) was engineered to contain a tumor specific E2F1 promoter driving an E1 gene deleted at the retinoblastoma protein binding site (“Δ24”). The fiber features a knob from serotype 3 for enhanced gene delivery to tumor cells. The virus was tested preclinically in vitro and in vivo and then 13 patients with solid tumors refractory to standard therapies were treated. Treatments were well tolerated and frequent tumor- and adenovirus-specific T-cell immune responses were seen. Overall, with regard to tumor marker or radiological responses, signs of antitumor efficacy were seen in 9/12 evaluable patients (75%). The radiological disease control rate with positron emission tomography was 83% while the response rate (including minor responses) was 50%. Tumor biopsies indicated accumulation of immunological cells, especially T-cells, to tumors after treatment. RNA expression analyses of tumors indicated immunological activation and metabolic changes secondary to virus replication.

Oncolytic immunotherapy with cytokine armed replication competent viruses is an emerging approach in cancer treatment. In a recent randomized trial, an increase in response rate was seen but the effect on overall survival is not known with any virus. To facilitate randomized trials, we performed a case–control study assessing the survival of 270 patients treated in an Advanced Therapy Access Program (ATAP), in comparison to matched concurrent controls from the same hospital. The overall survival of all virus treated patients was not increased over controls. However, when analysis was restricted to GMCSF-sensitive tumor types treated with GMSCF-coding viruses, a significant improvement in median survival was present (from 170 to 208 days, P = 0.0012, N = 148). An even larger difference was seen when analysis was restricted to good performance score patients (193 versus 292 days, P = 0.034, N = 90). The survival of ovarian cancer patients was especially promising as median survival nearly quadrupled (P = 0.0003, N = 37). These preliminary data lend support to initiation of randomized clinical trials with GMCSF-coding oncolytic adenoviruses.

After the landmark approval of T-VEC, oncolytic viruses are finding their way to the clinics. However, response rates have still room for improvement, and unfortunately there are currently no available markers to predict responses for oncolytic immunotherapy. Interleukin 8 (IL-8) production is upregulated in many cancers and it also connects to several pathways that have been shown to impair the efficacy of adenoviral immunotherapy. We studied the role of IL-8 in 103 cancer patients treated with oncolytic adenoviruses. We found high baseline serum IL-8 concentration to be independently associated with poor prognosis (p

Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing.

IntroductionAlthough a great majority of patients with cervical radiculopathy syndrome can successfully be treated non-operatively, a considerable proportion experience persistent symptoms, severe enough to require neurosurgical intervention. During the past decade, cervical spine procedures have increasingly been performed on an outpatient basis and retrospective database analyses have shown this to be feasible and safe. However, there are no randomised controlled studies comparing outpatient care with inpatient care, particularly with emphasis on the patients’ perception of symptom relief and their ability to return to normal daily activities and work.Methods and analysisThis is a prospective, randomised, controlled, parallel group non-inferiority trial comparing the traditional hospital surveillance (inpatient, patients staying in the hospital for 1–3 nights after surgery) with outpatient care (discharge on the day of the surgery, usually within 6–8 hours after procedure) in patients who have undergone anterior cervical decompression and fusion procedure. To determine whether early discharge (outpatient care) is non-inferior to inpatient care, we will randomise 104 patients to these two groups and follow them for 6 months using the Neck Disability Index (NDI) as the primary outcome. We expect that early discharge is not significantly worse than the current care in terms of change in NDI. Non-inferiority will be declared if the mean improvement for outpatient care is no worse than the mean improvement for inpatient care, by a margin of 17.3%. We hypothesise that a shorter hospital stay results in more rapid return to normal daily activities, shorter duration of sick leave and decreased secondary costs to healthcare system. Secondary outcomes in our study are arm pain and neck pain using the Numeric Rating Scale, operative success (Odom’s criteria), patient’s satisfaction to treatment, general quality of life (EQ-5D-5L), Work Ability Score, sickness absence days, return to previous leisure activities and complications.Ethics and disseminationThe study was approved by the institutional review board of the Helsinki and Uusimaa Hospital District on 6 June 2019 (1540/2019) and duly registered at ClinicalTrials.gov. We will disseminate the findings of this study through peer-reviewed publications and conference presentations.Trial registration numberNCT03979443.

At present, it is not possible to reliably identify patients who will benefit from oncolytic virus treatments. Conventional modalities such as computed tomography (CT), which measure tumor size, are unreliable owing to inflammation-induced tumor swelling. We hypothesized that magnetic resonance imaging (MRI) and spectroscopy (MRS) might be useful in this regard. However, little previous data exist and neither oncolytic adenovirus nor immunocompetent models have been assessed by MRS. Here, we provide evidence that in T2-weighted MRI a hypointense core area, consistent with coagulative necrosis, develops in immunocompetent Syrian hamster carcinomas that respond to oncolytic adenovirus treatment. The same phenomenon was observed in a neuroblastoma patient while he responded to the treatment. With relapse at a later stage, however, the tumor of this patient became moderately hyperintense. We found that MRS of taurine, choline and unsaturated fatty acids can be useful early indicators of response and provide detailed information about tumor growth and degeneration. In hamsters, calprotectin-positive inflammatory cells (heterophils and macrophages) were found in abundance; particularly surrounding necrotic areas in carcinomas and T cells were significantly increased in sarcomas, when these had been treated with a granulocyte-macrophage colony-stimulating factor-producing virus, suggesting a possible link between oncolysis, necrosis (seen as a hypointense core in MRI) and/or immune response. Our study indicates that both MRI and MRS could be useful in the estimation of oncolytic adenovirus efficacy at early time points after treatment.

Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.

Computed tomography (CT) is the most commonly used radiological response evaluation method in contemporary oncology. However, it may not be optimally suitable for assessment of oncolytic virus treatments because of paradoxical inflammatory tumor swellings, which result from virus treatments, particularly when viruses are armed with immunostimulatory molecules. Here we investigated the prognostic utility of CT and [(18)F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) in oncolytic virus treatments. We also investigated possible appearance of false-positive FDG signals in FDG-PET imaging of humans and hamsters treated with oncolytic adenoviruses. First, immunocompetent Syrian hamsters were treated with intratumoral adenovirus injections, tumor growth was followed up, and [(18)F]-FDG-uptake was quantitated with small animal PET/CT. Second, we describe a retrospective patient series, essentially 17 individual case reports, of advanced cancer patients treated with oncolytic adenoviruses in the context of an Advanced Therapy Access Program (ATAP) who underwent radiological response evaluation with both contrast-enhanced CT and FDG-PET. Third, we collected a retrospective case series of radiological response and survival data of 182 patients treated with oncolytic adenoviruses in ATAP to evaluate the prognostic reliability of CT and FDG-PET. Overall, responses in CT and FDG-PET correlated well with each other and were equally reliable as prognostic markers for long survival after oncolytic adenovirus treatment. Interestingly, we observed that new FDG-avid lymph nodes appearing in FDG-PET after virus treatments may represent inflammatory responses and therefore should not be interpreted as treatment failure in the absence of other signs or verification of disease progression. We also observed indications that FDG-PET might be more sensitive in detection of responses than tumor size.

The development of oncolytic viruses has recently made great progress towards being available to cancer patients. With the breakthrough into clinics, it is crucial to analyze the existing clinical experience and use it as a basis for treatment improvements. Here, we report clinical data from 290 patients treated with oncolytic adenovirus. Using clinical variables and treatment characteristics, we constructed statistical models with regard to treatment response and overall survival (OS). Additionally, we investigated effects of neutralizing antibodies, tumor burden, and peripheral blood leucocyte counts on these outcomes. We found the absence of liver metastases to correlate with an improved rate of disease control (P = 0.021). In multivariate evaluation, patients treated with viruses coding for immunostimulatory granulocyte macrophage colony-stimulating factor were linked to better prognosis (hazard ratio (HR) 0.378, P < 0.001), as well as women with any cancer type (HR 0.694, P = 0.017). In multivariate analysis for imaging response, patients treated via intraperitoneal injection were more likely to achieve disease control (odds ratio (OR) 3.246, P = 0.027). Patients with low neutrophil-to-lymphocyte ratio before treatment had significantly longer OS (P < 0.001). These findings could explain some of the variation seen in treatment outcomes after virotherapy. Furthermore, the results offer hypotheses for treatment optimization and patient selection in oncolytic adenovirus immunotherapy.

Oncolytic adenoviruses have been safe in clinical trials but the efficacy has been mostly limited. All published trials have been performed with serotype 5 based viruses. The expression level of the Ad5 receptor CAR may be variable in advanced tumors. In contrast, the Ad3 receptor remains unclear, but is known to be abundantly expressed in most tumors. Therefore, we hypothesized that a fully serotype 3 oncolytic adenovirus might be useful for treating cancer. Patients exposed to adenoviruses develop high titers of serotype-specific neutralizing antibodies, which might compromise re-administration. Thus, having different serotype oncolytic viruses available might facilitate repeated dosing in humans. Ad3-hTERT-E1A is a fully serotype 3 oncolytic adenovirus controlled by the promoter of the catalytic domain of human telomerase. It was effective in vitro on cell lines representing seven major cancer types, although low toxicity was seen in non-malignant cells. In vivo, the virus had anti-tumor efficacy in three different animal models. Although in vitro oncolysis mediated by Ad3-hTERT-E1A and wild-type Ad3 occurred more slowly than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls. Anti-tumor efficacy was retained in presence of neutralizing anti-Ad5 antibodies whereas Ad5 based controls were blocked. In summary, we report generation of a non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients, especially in the context of high anti-Ad5 neutralizing antibodies.

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.

Background Rapid clearance of adenoviruses from blood by macrophage lineage cells of the liver and spleen, and binding to platelets, hinder their successful systemic use for cancer gene therapy. Vitamin K dependent coagulation factors are important mediators for the adenovirus liver tropism. Here we aim to determine the effects of coagulation factor, thrombocyte and liver macrophage (Kupffer cell) ablation on biodistribution of serotype 5 adenoviruses in mice with orthotopic breast tumors. Methods Prior to intravenous injection of adenoviruses, mice bearing orthotopic breast tumors were pretreated with warfarin to inhibit vitamin K dependent coagulation factor synthesis, an anti-platelet antibody causing thrombocytopenia or an inhibitor of the Kupffer cell scavenger receptor or their combination. Virus availability in blood after injection was determined from blood samples and transgene expression in tissues analyzed 72 hours afterwards with In Vivo Imaging and luciferase assays. Results Warfarin pretreatment reduced gene delivery to liver, spleen and lung. Kupffer cell ablation increased persistence of adenoviruses in blood but didn't affect biodistribution significantly. Depletion of Kupffer cells combined with thrombocytopenia doubled the systemic gene delivery of 5/3 chimeric adenovirus to tumors (p < 0.05). Triple ablation of platelets, Kupffer cells and coagulation factors increased the tumor to liver ratio of systemic adenovirus gene delivery by 81% (p < 0.05). Conclusions Depletion of coagulation factors can reduce transduction of liver, spleen and lung. Kupffer cell depletion is the most feasible method of increasing amount adenovirus in systemic blood flow and in combination with ablation of thrombocytes can increase the transduction of adenovirus to tumors. Copyright © 2009 John Wiley & Sons, Ltd.

Meeting abstracts After years of development, oncolytic viruses are nearing a breakthrough into clinical use. In order to facilitate the implementation of this new form of immunotherapy, all existing clinical experience must be utilized to optimize treatment strategies, improve patient selection

Breast cancer is a heterogeneous disease, characterized by several distinct biological subtypes, among which triple-negative breast cancer (TNBC) is one associated with a poor prognosis. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules to boost virus triggered antitumoral immune responses. Cyclophosphamide (CP) is a chemotherapy drug that is associated with cytotoxicity and immunosuppression at higher doses, whereas immunostimulatory and anti-angiogenic properties are observed at low continuous dosage. Therefore, the combination of oncolytic immuno-virotherapy with low-dose CP is an appealing approach. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a TNBC cell line and in vivo in an orthotopic xenograft mouse model, in combination with low-dose CP or its main active metabolite 4-hydroperoxycyclophosphamide (4-HP-CP). Furthermore, we summarized the breast cancer-specific human data on this virus from the Advanced Therapy Access Program (ATAP). Low-dose CP increased the efficacy of Ad5/3-D24-GMCSF in vitro and in a TNBC mouse model. In ATAP, treatments appeared safe and well-tolerated. Thirteen out of 16 breast cancer patients treated were evaluable for possible benefits with modified RECIST 1.1 criteria: 1 patient had a minor response, 2 had stable disease (SD), and 10 had progressive disease (PD). One patient is alive at 1,771 d after treatment. Ad5/3-D24-GMCSF in combination with low-dose CP showed promising efficacy in preclinical studies and possible antitumor activity in breast cancer patients refractory to other forms of therapy. This preliminary data supports continuing the clinical development of oncolytic adenoviruses for treatment of breast cancer, including TNBC.

Purpose: Multiple injections of oncolytic adenovirus could enhance immunologic response. In the first part of this article, the focus was on immunologic aspects. Sixty patients previously naïve to oncolytic virus and who had white blood cells available were treated. Thirty-nine of 60 were assessed after a single virus administration, whereas 21 of 60 received a “serial treatment” consisting of three injections within 10 weeks. In the second part, we focused on 115 patients treated with a granulocyte macrophage colony-stimulating factor (GM–CSF)–coding capsid chimeric adenovirus, CGTG-102. Results: Following serial treatment, both increase and decrease in antitumor T cells in blood were seen more frequently, findings which are compatible with induction of T-cell immunity and trafficking of T cells to tumors, respectively. Safety was good in both groups. In 115 patients treated with CGTG-102 (Ad5/3-D24-GMCSF), median overall survival was 111 days following single and 277 days after serial treatment in nonrandomized comparison. Switching the virus capsid for avoiding neutralizing antibodies in a serial treatment featuring three different viruses did not impact safety or efficacy. A correlation between antiviral and antitumor T cells was seen (P = 0.001), suggesting that viral oncolysis can result in epitope spreading and breaking of tumor-associated immunologic tolerance. Alternatively, some patients may be more susceptible to induction of T-cell immunity and/or trafficking. Conclusions: These results provide the first human data linking antiviral immunity with antitumor immunity, implying that oncolytic viruses could have an important role in cancer immunotherapy. Clin Cancer Res; 19(10); 2734–44. ©2013 AACR.

Oncolytic immunotherapy has taken significant steps towards clinical availability for cancer patients, as the FDA recently approved Imlygic (T-Vec) for the treatment of melanoma. Despite the several oncolytic virus trials, there are no known biomarkers which could predict the treatment outcome of oncolytic virotherapy. Therefore, previous clinical experiences should be utilized as guidance for further treatment optimization and patient selection. The patients analyzed consisted of 246 patients treated with oncolytic adenovirus as a part of the Advanced Therapy Access Program which was ongoing in Helsinki 2007-2012. Taken together with the increasing understanding that immunological factors might determine the efficacy of oncolytic virotherapy, we studied whether neutralizing antibodies and peripheral blood cell counts would have prognostic and/or predictive significance in the context of oncolytic adenovirus treatments. Additionally, tumor burden was assessed both in terms of primary tumor and metastases. We observed presence of neutralizing antibodies before treatment to be correlated with shorter overall survival (p=0.028). Blood cell counts analyses revealed that patients who had high neutrophil to lymphocyte ratio at baseline had significantly shorter overall survival (p

Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid-modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre-existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six-fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach.

Adenovirus is one of the most commonly used vectors for gene therapy and two products have already been approved for treatment of cancer in China (Gendicine(R) and Oncorine(R)). An intriguing aspect of oncolytic adenoviruses is that by their very nature they potently stimulate multiple arms of the immune system. Thus, combined tumor killing via oncolysis and inherent immunostimulatory properties in fact make these viruses in situ tumor vaccines. When further engineered to express cytokines, chemokines, tumor-associated antigens, or other immunomodulatory elements, they have been shown in various preclinical models to induce antigen-specific effector and memory responses, resulting both in full therapeutic cures and even induction of life-long tumor immunity. Here, we review the state of the art of oncolytic adenovirus, in the context of their capability to stimulate innate and adaptive arms of the immune system and finally how we can modify these viruses to direct the immune response toward cancer.

Patients with advanced solid tumors refractory to and progressing after conventional therapies were treated with three different regimens of low-dose cyclophosphamide (CP) in combination with oncolytic adenovirus. CP was given with oral metronomic dosing (50 mg/day, N = 21), intravenously (single 1,000 mg dose, N = 7) or both (N = 7). Virus was injected intratumorally. Controls (N = 8) received virus without CP. Treatments were well tolerated and safe regardless of schedule. Antibody formation and virus replication were not affected by CP. Metronomic CP (oral and oral + intravenous schedules) decreased regulatory T cells (Tregs) without compromising induction of antitumor or antiviral T-cell responses. Oncolytic adenovirus given together with metronomic CP increased cytotoxic T cells and induced Th1 type immunity on a systemic level in most patients. All CP regimens resulted in higher rates of disease control than virus only (all P < 0.0001) and the best progression-free (PFS) and overall survival (OS) was seen in the oral + intravenous group. One year PFS and OS were 53 and 42% (P = 0.0016 and P < 0.02 versus virus only), respectively, both which are unusually high for chemotherapy refractory patients. We conclude that low-dose CP results in immunological effects appealing for oncolytic virotherapy. While these first-in-human data suggest good safety, intriguing efficacy and extended survival, the results should be confirmed in a randomized trial.

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as ipilimumab or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models in combination with low-dose cyclophosphamide, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. Four out of nine melanoma patients treated in ATAP were evaluable for possible therapy benefit with modified Response Evaluation Criteria In Solid Tumors (RECIST): one patient had minor response, two had stable disease, and one had progressive disease. Treatments appeared safe and well-tolerated. Two patients were alive at 559 and 1501 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma. Citation Format: Simona Bramante, Johanna K. Kaufmann, Ville Veckman, Dirk M. Nettelbeck, Otto Hemminki, Ilkka Liikanen, Lotta Vassilev, Vincenzo Cerullo, Minna Oksanen, Raita Heiskanen, Timo Joensuu, Anna Kanerva, Sari Pesonen, Markus Vähä-Koskela, Anniina Koski, Akseli Hemminki. Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B51.

Breast cancer is a heterogeneous disease, characterized by several distinct biological subtypes associated with specifical biological behavior and different clinical outcomes. Triple-negative breast cancer (TNBC) is defined as estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and lacking overexpression of human epidermal growth factor receptor 2 (HER2). Among all the breast cancer subtypes, TNBC (10-15% of breast cancers) is associated with a worse prognosis.Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Cyclophosphamide (CP) is a chemotherapy drug that works by slowing or stopping cell growth. At higher doses, CP is associated with increased cytotoxicity and immunosuppression, while at low continuous dosage it shows immunostimulatory and antiangiogenic properties. Therefore, the combination of oncolytic immunovirotherapy with low-dose CP is an appealing approach.We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a TNBC cell line and animal models, in combination with low-dose CP or its main active metabolite 4-hydroxycyclophosphamide (4-HP-CP). Furthermore, we summarized the breast cancer-specific human data from the Advanced Therapy Access Program (ATAP).Low-dose CP effectively increased the efficacy of Ad5/3-D24-GMCSF in vitro and in a TNBC xenograft mouse model. In ATAP, treatments appeared safe and well-tolerated. Fourteen out of sixteen breast cancer patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, four had stable disease, and nine had progressive disease. One patient was alive at 941 days after treatment.Ad5/3-D24-GMCSF in combination with low-dose CP showed promising efficacy in preclinical studies and possible antitumor activity in breast cancer patients refractory to other forms of therapy. This preliminary data supports continuing the clinical development of oncolytic adenoviruses for treatment of breast cancer, including TNBC.

With the emergence of effective immunotherapeutics, which nevertheless harbor the potential for toxicity and are expensive to use, biomarkers are urgently needed for identification of cancer patients who respond to treatment. In this clinical-epidemiological study of 202 cancer patients treated with oncolytic adenoviruses, we address the biomarker value of serum high-mobility group box 1 (HMGB1) protein. Overall survival and imaging responses were studied as primary endpoints and adjusted for confounding factors in two multivariate analyses (Cox and logistic regression). Mechanistic studies included assessment of circulating tumor-specific T-cells by ELISPOT, virus replication by quantitative PCR, and inflammatory cytokines by cytometric bead array. Patients with low HMGB1 baseline levels (below median concentration) showed significantly improved survival (p = 0.008, Log-Rank test) and radiological disease control rate (49.2% vs. 30.0%, p = 0.038, χ2 test) as compared to high-baseline patients. In multivariate analyses, the low HMGB1 baseline status was a strong prognostic (HR 0.638, 95% CI 0.462–0.881) and the best predictive factor for disease control (OR 2.618, 95% CI 1.004–6.827). Indicative of an immune-mediated mechanism, antitumor T-cell activity in blood and response to immunogenic-transgene coding viruses associated with improved outcome only in HMGB1-low patients. Our results suggest that serum HMGB1 baseline is a useful prognostic and predictive biomarker for oncolytic immunotherapy with adenoviruses, setting the stage for prospective clinical studies.

Tumor cell autophagy appears useful for cancer therapeutics due to immunogenic cell death and possible induction of antitumor immunity. Oncolytic adenoviruses and alkylating chemotherapeutic temozolomide have been shown to induce autophagic cell death preclinically. We studied safety, efficacy and immunological effects of oncolytic adenovirus combined with low-dose pulse of temozolomide. Metronomic low-dose cyclophosphamide was added to treatments to selectively reduce regulatory T-cells. We assessed the combination of oncolytic adenovirus with low-dose temozolomide and cyclophosphamide in breast and prostate cancer cells in vitro. Immunogenicity of cell death was studied by calreticulin exposure, adenosine triphosphate release, and nuclear protein high-mobility group box-1 (HMGB1) secretion assays. Efficacy was further tested in a prostate cancer xenograft mouse model, and tumors were assessed for autophagy by electron microscopy and LC3-immunohistochemistry. Furthermore, 41 combination treatments were given to 17 chemotherapy-refractory cancer patients in the context of an advanced therapy access program. The primary end-point was safety. In addition, autophagy assays, immunological analyses, and efficacy parameters were studied. Combination therapy resulted in increased cytotoxicity and immunogenic cell death. Autophagy induction was associated with enhanced tumor-growth inhibition in combination-treated prostate tumors (P Low-dose pulse of temozolomide and cyclophosphamide in combination with oncolytic adenovirus increased autophagy, elicited antitumor T-cell activations, and showed promising safety and efficacy. Our results support the hypothesis that oncolysis and chemotherapy mediated autophagy might lead to antitumor immunity. Oncolytic adenovirus together with autophagy-inducing temozolomide is an attractive combination for future clinical studies. Citation Format: Ilkka Liikanen, Laura Ahtiainen, Mari L.M, Hirvinen, Simona Barmante, Vincenzo Cerullo, Petri Nokisalmi, Otto Hemminki, Iulia Diaconu, Sari Pesonen, Anniina Koski, Lotta Kangasniemi, Saila K. Pesonen, Minna Oksanen, Fang Zhao, Leena Laasonen, Kaarina Partanen, Timo Joensuu, Anna Kanerva, Akseli Hemminki. Oncolytic adenovirus with low-dose temozolomide induces autophagy and antitumor immune responses preclinically and in cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2013-3304