Integrating qualitative interviews in drug development and the use of qualitative evidence in product labelling and health technology assessments: a review.

Objective: Including qualitative research in clinical trial design is an innovative approach to understanding patients' perspective and incorporate the patient's voice in all stages of drug development and evaluation. This review aims to explore current practices, lessons learned from the literature, as well as how qualitative interviews are considered by health authorities for marketing authorization and reimbursement. Methods: A targeted literature review of Medline and Embase databases was conducted in February 2022 to identify publications on qualitative methods embedded in clinical trial of pharmaceutical products. An additional search of guidelines and labeling claims of approved products regarding qualitative research was performed in various sources of grey literature. Results: From the 24 publications and nine documents reviewed, we identified the research questions addressed with qualitative methods during clinical trials (e.g., change in quality of life, symptoms assessment, treatment benefit), preferred data collection methods (e.g., interviews), and data collection points (e.g., baseline and exit interviews). Moreover, the data from labels and HTAs demonstrate that qualitative data can play an important role in approval processes. Conclusion: The use of in-trial interviews is still emerging and is not yet common practice. Although the industry, scientific community, regulatory agencies and HTAs are showing an increasing interest in the use of evidence generated via in-trial interviews, guidance from regulators and HTAs would be helpful. Developing new methods and technologies to address the common challenges for such interviews is key to progress.

Key considerations to reduce or address respondent burden in patient-reported outcome (PRO) data collection.

Patient-reported outcomes (PROs) are used in clinical trials to provide evidence of the benefits and risks of interventions from a patient perspective and to inform regulatory decisions and health policy. The collection of PROs in routine practice can facilitate monitoring of patient symptoms; identification of unmet needs; prioritisation and/or tailoring of treatment to the needs of individual patients and inform value-based healthcare initiatives. However, respondent burden needs to be carefully considered and addressed to avoid high rates of missing data and poor reporting of PRO results, which may lead to poor quality data for regulatory decision making and/or clinical care.

Meganuclease-mediated virus self-cleavage facilitates tumor-specific virus replication.

Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome. We observed that virus replication and cell lysis was effectively impaired in p53-normal cells, but not in p53-dysfunctional tumor cells. I-Sce I activity led to effective intracellular processing of viral DNA as confirmed by detection of specific cleavage products. Virus disruption did not interfere with E1A levels indicating that reduction of functional virus genomes was the predominant cause for conditional replication. Consequently, tumor-specific replication was further enhanced when E1A expression was additionally inhibited by targeted transcriptional repression. Finally, we demonstrated p53-dependent oncolysis by I-Sce I-expressing viruses in vitro and in vivo, and demonstrated effective inhibition of tumor growth. In summary, meganuclease-mediated virus cleavage represents a promising approach to provide oncolytic viruses with attractive safety profiles.

Virus-induced tumor inflammation facilitates effective DC cancer immunotherapy in a Treg-dependent manner in mice.

Vaccination using DCs pulsed with tumor lysates or specific tumor-associated peptides has so far yielded limited clinical success for cancer treatment, due mainly to the low immunogenicity of tumor-associated antigens. In this study, we have identified intratumoral virus-induced inflammation as a precondition for effective antitumor DC vaccination in mice. Administration of a tumor-targeted DC vaccine during ongoing virus-induced tumor inflammation, a regimen referred to as oncolysis-assisted DC vaccination (ODC), elicited potent antitumoral CD8+ T cell responses. This potent effect was not replicated by TLR activation outside the context of viral infection. ODC-elicited immune responses mediated marked tumor regression and successful eradication of preestablished lung colonies, an essential prerequisite for potentially treating metastatic cancers. Unexpectedly, depletion of Tregs during ODC did not enhance therapeutic efficacy; rather, it abrogated antitumor cytotoxicity. This phenomenon could be attributed to a compensatory induction of myeloid-derived suppressor cells in Treg-depleted and thus vigorously inflamed tumors, which prevented ODC-mediated immune responses. Consequently, Tregs are not only general suppressors of immune responses, but are essential for the therapeutic success of multimodal and temporally fine-adjusted vaccination strategies. Our results highlight tumor-targeting, replication-competent viruses as attractive tools for eliciting effective antitumor responses upon DC vaccination.

Antitumoural immunity by virus-mediated immunogenic apoptosis inhibits metastatic growth of hepatocellular carcinoma.

BACKGROUND AND AIMS: Viral infection of a dying cell dictates the immune response against intracellular antigens, suggesting that virotherapy may be an effective tool to induce immunogenic cell death during systemic cancer treatment. Since viruses and proteasome inhibitors both induce accumulation of misfolded proteins, endoplasmic reticulum (ER) stress and immune responses during treatment of hepatocellular carcinoma (HCC) with bortezomib and the tumour-specifically replicating virus hTert-Ad (human telomerase reverse transcriptase promoter-regulated adenovirus) were investigated. METHODS: Unfolded protein response (UPR) pathways and ER stress-mediated apoptosis were investigated by western blots, caspase-3 assays, 4',6-diamidino-2-phenylindole (DAPI) and Annexin V staining in HCC cells following hTert-Ad/bortezomib treatment. Oncolysis was assessed in subcutaneous HCC mouse models. Antiviral/antitumoural immune responses were characterised in immunocompetent HCC mouse models by ELISA, ELISpot assays and pentamer staining. Systemic efficacy of antitumoural immunity was investigated by determination of lung metastases burden. RESULTS: Bortezomib and hTert-Ad trigger complementary UPR pathways but negatively interfere with important recovery checkpoints, resulting in enhanced apoptosis of HCC cells in vitro and improved oncolysis in vivo. In immunocompetent mice, bortezomib inhibited antiviral immune responses, whereas ER stress-induced apoptosis of infected HCC resulted in caspase-dependent triggering of antitumoural immunity. In therapeutic settings in immunocompetent, but not in immunodeficient or CD8-depleted mice, virotherapy-induced antitumoural immunity efficiently inhibited outgrowth of non-infected lung metastases. Immunotherapeutic efficacy could be significantly improved by bortezomib in experiments with low viral doses. CONCLUSION: Proteasome inhibition during virotherapy disrupts the UPR, leading to enhanced ER stress-induced apoptosis, improved local oncolysis and antitumoural immunity. The results suggest that combining intratumoural virotherapy with adjuvant systemic therapies, which specifically support the function of the virotherapy as an antitumoural vaccine, is a promising immunotherapeutic strategy against HCC.

Selectivity of oncolytic viral replication prevents antiviral immune response and toxicity, but does not improve antitumoral immunity.

Oncolytic infection elicits antitumoral immunity, but the impact of tumor-selective replication on the balance between antiviral and antitumoral immune responses has not yet been investigated. To address this question, we constructed the highly tumor-selective adenovirus Ad-p53T whose replication in target tumor cells is governed by aberrant telomerase activity and transcriptional p53 dysfunction. Telomerase-dependent or nonselective adenoviruses were constructed as isogenic controls. Following infection of mice with the nonselective adenovirus, viral DNA and mRNA levels correlated with strong stimulation of innate immune response genes and severe liver toxicity, whereas telomerase-/p53-specific replication did not trigger innate immunity and prevented liver damage. Compared to telomerase-dependent or unselective viral replication, telomerase-/p53-specific virotherapy significantly decreased antiviral CD8-specific immune responses and antiviral cytotoxicity in vivo. Consistent with our hypothesis, telomerase-selective replication led to intermediate results in these experiments. Remarkably, all viruses efficiently lysed tumors and induced a therapeutically effective tumor-directed CD8 cytotoxicity. In immunocompetent mice with extended lung metastases burden, treatment of subcutaneous primary tumors with Ad-p53T significantly prolonged survival by inhibition of lung metastases, whereas unselective viral replication resulted in death by liver failure. In summary, the degree of tumor selectivity of viral replication marginally influences antitumoral immune responses, but is a major determinant of antivector immunity and systemic toxicity.

p53-dependent antiviral RNA-interference facilitates tumor-selective viral replication.

RNA-interference (RNAi) is a potent tool for specific gene silencing. In this study, we developed an adenovirus for conditional replication in p53-dysfunctional tumor cells that uses p53-selective expression of a microRNA-network directed against essential adenoviral genes. Compared to a control virus that expressed a scrambled microRNA-network, antiviral RNAi selectively attenuated viral replication in cells with transcriptionally active p53, but not in p53-dysfunctional tumor cells where both viruses replicated equivalently. Since these results were confirmed by an in vivo comparison of both viruses after infection of p53-knockout and normal mice, we could demonstrate that attenuated replication was indeed a result of p53-selective exhibition of antiviral RNAi. Addressing the therapeutic applicability, we could show that the application of RNAi-controlled virus efficiently lysed p53-dysfunctional tumors in vitro and in vivo but resulted in drastically reduced load of virus-DNA in the liver of treated mice. We have generated a broadly applicable adenovirus for selective destruction of p53-dysfunctional tumors and thereby demonstrate that virus-encoded RNAi-networks represent an efficient and versatile tool to modify viral functions. RNAi-networks can be applied to all transcriptionally regulated DNA-viruses to remodulate viral tropism and thus provide means to generate specifically replicating vectors for clinical applications.

The human topoisomerase I damage response plays a role in apoptosis.

Previous studies have shown that human topoisomerase I cleavage complexes form as a response to various DNA damages in vivo, the so called human topoisomerase I "damage response". It was suggested that this damage response may play a role in DNA repair as well as in apoptosis, but only very few investigations have been done and the significance of the damage response still remains unclear. Here we demonstrate that human topoisomerase I cleavage complexes induced by high doses of UV irradiation are highly stable for up to 48 h. Furthermore, we show that human topoisomerase I cleavage complexes correlate with apoptosis. However, at low UV doses the cleavage complex level was very low and the complexes were repaired. Surprisingly, we found that high levels of stable cleavage complexes were not only found in UV-irradiated cells but also in untreated cells that underwent apoptosis. A possible role of human topoisomerase I in apoptosis is discussed.