With few exceptions, current chemotherapy and radiotherapy protocols only obtain a slightly prolonged survival with severe adverse effects in patients with advanced solid tumors. In particular, most solid malignancies not amenable to radical surgery still carry a dismal prognosis, which unfortunately is also the case for relapsing disease after surgery. Even though targeted therapies obtained good results, clinical experience showed that tumors eventually develop resistance. On the other hand, earlier attempts of cancer immunotherapy failed to show consistent efficacy. More recently, a deeper knowledge of immunosuppression in the tumor microenvironment (TME) allowed the development of effective drugs: in particular, monoclonal antibodies targeting the so-called immune checkpoint molecules yielded striking and lasting effects in some tumors. Unfortunately, these monoclonal antibodies are not effective in a majority of patients and are ineffective in several solid malignancies. Furthermore, due to their mechanism of action, checkpoint inhibitors often elicit autoimmune-like disease. The use of viruses as oncolytic agents (OVs) was considered in the past, while only recently OVs revealed a connection with immunotherapy. However, their antitumoral potential has remained largely unexplored, due to safety concerns and some limitations in the techniques to manipulate viruses. OV research was recently revived by a better knowledge of viral/cancer biology and advances in the methodologies to delete virulence/immune-escape related genes from even complex viral genomes or “to arm” OVs with appropriate transgenes. Recently, the first oncolytic virus, the HSV-1 based Talimogene Laherparepvec (T-VEC), was approved for the treatment of non-resectable melanoma in USA and Europe. OVs have the potential to become powerful agents of cancer immune and gene therapy. Indeed, in addition to their selective killing activity, they can act as versatile gene expression platforms for the delivery of therapeutic genes. This is particularly true for viruses with a large DNA genome, that can be manipulated to address the multiple immunosuppressive features of the TME. This review will focus on the open issues, on the most promising lines of research in the OV field and, more in general, on how OVs could be improved to achieve real clinical breakthroughs in cancers that are usually difficult to treat by immunotherapy.

Oncolytic viruses (OVs) are anti-cancer therapeutics combining the selective killing of cancer cells with the triggering of an anti-tumoral immune response. The latter effect can be improved by arming OVs with immunomodulatory factors. Due to the heterogeneity of cancer and the tumor microenvironment, it is anticipated that strategies based on the co-expression of multiple therapeutic molecules that interfere with different features of the target malignancy will be more effective than mono-therapies. Here, we show that (i) the simultaneous expression of different proteins in triple-negative breast cancer (TNBC) cells can be achieved through their infection with a combination of OVs based on herpes simplex virus type 1 (oHSV1), each encoding a single transgene. (ii) The level of expressed proteins is dependent on the number of infectious viral particles utilized to challenge tumor cells. (iii) All recombinant viruses exhibited comparable efficacy in the killing of TNBC cells in single and multiple infections and showed similar kinetics of replication. Overall, our results suggest that a strategy based on co-infection with a panel of oHSV1s may represent a promising combinatorial therapeutic approach for TNBC, as well as for other types of solid tumors, that merits further investigation in more advanced in vitro and in vivo models.

Mycoplasma pneumoniae is a significant causative agent of atypical pneumonia in both children and adults. Timely and accurate diagnosis is crucial for appropriate patient management. Conventional methods for detecting M. pneumoniae, such as culture and serology, exhibit several limitations regarding sensitivity, specificity, and turnaround time. In contrast, real-time PCR is considered the most reliable, rapid, and sensitive technique for the diagnosis of M. pneumoniae infection. In this study, we adapted and validated an in-house real-time PCR assay for use on the fully automated Panther Fusion^® System. The validation process included two artificial samples, five external quality controls, and sixty-two patient samples. We evaluated the performance in terms of precision, sensitivity, linearity, and analytical sensitivity, comparing it to the original in-house assay. The Panther Fusion^® System demonstrated a broad dynamic range (16–1.6 × 10⁷ copies/reaction), a robust correlation (94%) with the in-house assay, and comparable sensitivity (46 copies/mL vs. 25 copies/mL). The concordance between the in-house real-time PCR and the Panther Fusion^® System was 100% for both clinical samples and external quality controls. The adaptation of the test to the Panther Fusion^® System enabled the inclusion of M. pneumoniae among the pathogens monitored for respiratory infection surveillance. Throughout 2024, we analyzed 2567 samples, with a peak positivity rate of 38% observed in August. These findings underscore the significance of employing the M. pneumoniae diagnostic assay on the Panther Fusion^® System which proves valuable for the detection of M. pneumoniae infections. This platform offers the advantages of increased automation and greater throughput potential compared to other platforms, enhancing the efficiency of respiratory pathogen detection in clinical settings.