Luigi Pasini

Background: By examining the B-cell receptor (BCR) repertoire of metastatic melanoma (MM) patients with favorable treatment outcomes, it is now possible to identify unique patterns of immune responses, with the potential of discovering novel antitumor antibodies.Methods: Here, we isolated CD27-positive circulating memory B cells from non responders, partial responders, and complete responders MM patients to first-line therapy with anti-PD-1 immune checkpoint inhibitor (ICI) nivolumab, to perform a BCR repertoire sequencing analysis. We looked for complementarity-determining region 3 (CDR3) sequences that were enriched (de novo formed) following ICI treatment. Fully-human immunoglobulins were then produced in Expi293F™ cells using CDR3-sequencing information and tested for specificity and sensitivity on different MM cell lines and patient-derived xenograft cells by flow cytometry and by immunohistochemistry on human tissue microarrays.Results: As a result of immunotherapy stimulation in responder patients, we observed that some CDR3 clonotypes have emerged de novo. Among the nine candidate antibodies we assessed, two antibodies exhibited encouraging tumor-targeting properties, although they also showed a degree of cross-reactivity with normal skin and melanocytes.Conclusions: Although our study is based on a limited number of individuals, our observations indicate that it may be possible to further investigate the human response to immunotherapy for the identification of rare mature B clonotypes targeting plasma membrane antigens on tumor cells. These preliminary findings could contribute to the future development of fully human-compatible immunotherapies, pending additional validation and in vivo studies.

Over the past decades, significant progress has been made in the understanding of non-small cell lung cancer (NSCLC) biology and tumor progression mechanisms, resulting in the development of novel strategies for early detection and wide-ranging care approaches. Since their introduction, over 20 years ago, targeted therapies with tyrosine kinase inhibitors (TKIs) have revolutionized the treatment landscape for NSCLC. Nowadays, targeted therapies remain the gold standard for many patients, but still they suffer from many adverse effects, including unexpected toxicity and intrinsic acquired resistance mutations, which lead to relapse. The adoption of immune checkpoint inhibitors (ICIs) in 2015, has offered exceptional survival benefits for patients without targetable alterations. Despite this notable progress, challenges remain, as not all patients respond favorably to ICIs, and resistance to therapy can develop over time. A crucial factor influencing clinical response to immunotherapy is the tumor microenvironment (TME). The TME is pivotal in orchestrating the interactions between neoplastic cells and the immune system, influencing tumor growth and treatment outcomes. In this review, we discuss how the understanding of this intricate relationship is crucial for the success of immunotherapy and survey the current state of immunotherapy intervention, with a focus on forthcoming and promising chimeric antigen receptor (CAR) T cell therapies in NSCLC. The TME sets major obstacles for CAR-T therapies, creating conditions that suppress the immune response, inducing T cell exhaustion. To enhance treatment efficacy, specific efforts associated with CAR-T cell therapy in NSCLC, should definitely focus TME-related immunosuppression and antigen escape mechanisms, by combining CAR-T cells with immune checkpoint blockades.

Recent times have witnessed remarkable progress in cancer immunotherapy, drastically changing the cancer treatment landscape. Among the various immunotherapeutic approaches, adoptive cell therapy (ACT), particularly chimeric antigen receptor (CAR) T cell therapy, has emerged as a promising strategy to tackle cancer. CAR-T cells are genetically engineered T cells with synthetic receptors capable of recognising and targeting tumour-specific or tumour-associated antigens. By leveraging the intrinsic cytotoxicity of T cells and enhancing their tumour-targeting specificity, CAR-T cell therapy holds immense potential in achieving long-term remission for cancer patients. However, challenges such as antigen escape and cytokine release syndrome underscore the need for the continued optimisation and refinement of CAR-T cell therapy. Here, we report on the challenges of CAR-T cell therapies and on the efforts focused on innovative CAR design, on diverse therapeutic strategies, and on future directions for this emerging and fast-growing field. The review highlights the significant advances and changes in CAR-T cell therapy, focusing on the design and function of CAR constructs, systematically categorising the different CARs based on their structures and concepts to guide researchers interested in ACT through an ever-changing and complex scenario. UNIVERSAL CARs, engineered to recognise multiple tumour antigens simultaneously, DUAL CARs, and SUPRA CARs are some of the most advanced instances. Non-molecular variant categories including CARs capable of secreting enzymes, such as catalase to reduce oxidative stress in situ, and heparanase to promote infiltration by degrading the extracellular matrix, are also explained. Additionally, we report on CARs influenced or activated by external stimuli like light, heat, oxygen, or nanomaterials. Those strategies and improved CAR constructs in combination with further genetic engineering through CRISPR/Cas9- and TALEN-based approaches for genome editing will pave the way for successful clinical applications that today are just starting to scratch the surface. The frontier lies in bringing those approaches into clinical assessment, aiming for more regulated, safer, and effective CAR-T therapies for cancer patients.

Lung cancer (LC) is the deadliest malignancy worldwide. In an operable stage I–III patient setting, the detection of minimal residual disease (MRD) after curative treatment could identify patients at higher risk of relapse. In this context, the study of circulating tumor DNA (ctDNA) is emerging as a useful tool to identify patients who could benefit from an adjuvant treatment, and patients who could avoid adverse events related to a more aggressive clinical management. On the other hand, ctDNA profiling presents technical, biological and standardization challenges before entering clinical practice as a decisional tool. In this paper, we review the latest advances regarding the role of ctDNA in identifying MRD and in predicting patients’ prognosis, with a particular focus on clinical trials investigating the potential of ctDNA, the technical challenges to address and the biological parameters that influence the MRD detection.

In recent years, circulating extracellular miRNAs have emerged as a useful tool for the molecular characterization and study of tumors’ biological functions. However, the high heterogeneity in sample processing, isolation of circulating fraction, RNA extraction, and sequencing hamper the reproducibility and the introduction of these biomarkers in clinical practice. In this paper, we compare the content and the performance of miRNA sequencing in plasma-derived samples processed with different isolation protocols. We tested three different fractions of miRNA from healthy-donor human blood: whole plasma (WP), free-circulating (FC) and EV-associated, isolated by either column (ccEV) or size exclusion chromatography (secEV) miRNAs. An additional cohort of 18 lung cancer patients was analyzed. Protein profiles of ccEV and secEV were compared and miRNA expression profiles were assessed through sequencing. Slight differences were found between ccEV and secEV expressions of typical EV markers. Conversely, sequencing performance and the mirnome profile varied between RNA extracted using different isolation methods. Sequencing performance was better in FC samples. Higher varieties of miRNAs were identified in WP and FC with respect to ccEV and secEV. Analysis of free-circulating and EV-associated miRNA profiles in lung cancer patients demonstrated the reliability of the biomarkers identifiable on plasma with these approaches.

Non-Small-Cell Lung Cancer (NSCLC) is the primary cause of cancer-related death worldwide. Oncogene-addicted patients usually benefit from targeted therapy, but primary and acquired resistance mechanisms inevitably occur. Tumor protein 53 (TP53) gene is the most frequently mutated gene in cancer, including NSCLC. TP53 mutations are able to induce carcinogenesis, tumor development and resistance to therapy, influencing patient prognosis and responsiveness to therapy. TP53 mutants present in different forms, suggesting that different gene alterations confer specific acquired protein functions. In recent years, many associations between different TP53 mutations and responses to Epidermal Growth Factor Receptor (EGFR) targeted therapy in NSCLC patients have been found. In this review, we discuss the current landscape concerning the role of TP53 mutants to guide primary and acquired resistance to Tyrosine-Kinase Inhibitors (TKIs) EGFR-directed, investigating the possible mechanisms of TP53 mutants within the cellular compartments. We also discuss the role of the TP53 mutations in predicting the response to targeted therapy with EGFR-TKIs, as a possible biomarker to guide patient stratification for treatment.

Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations.

Very few data are reported in the literature on the association between elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and prognosis in advanced colorectal cancer. Moreover, there is no information available in relation to the response to antiangiogenic treatment. We analyzed EMAST and vascular endothelial growth factor-B (VEGF-B) microsatellite status, together with standard microsatellite instability (MSI), in relation to prognosis in 141 patients with metastatic colorectal cancer (mCRC) treated with chemotherapy (CT) alone (n = 51) or chemotherapy with bevacizumab (B) (CT + B; n = 90). High MSI (MSI-H) was detected in 3% of patients and was associated with progression-free survival (PFS; p = 0.005) and overall survival (OS; p < 0.0001). A total of 8% of cases showed EMAST instability, which was associated with worse PFS (p = 0.0006) and OS (p < 0.0001) in patients treated with CT + B. A total of 24.2% of patients showed VEGF-B instability associated with poorer outcome in (p = 0.005) in the CT arm. In conclusion, our analysis indicated that EMAST instability is associated with worse prognosis, particularly evident in patients receiving CT + B.

Targeted and immunological therapies have become the gold standard for a large portion of non-small cell lung cancer (NSCLC) patients by improving significantly clinical prognosis. However, resistance mechanisms inevitably develop after a first response, and almost all patients undergo progression. The knowledge of such a resistance mechanism is crucial to improving the efficacy of therapies. So far, monitoring therapy responses through liquid biopsy has been carried out mainly in terms of circulating tumor (ctDNA) analysis. However, other particles of tumor origin, such as extracellular vehicles (EVs) represent an emerging tool for the studying and monitoring of resistance mechanisms. EVs are now considered to be ubiquitous mediators of cell-to-cell communication, allowing cells to exchange biologically active cargoes that vary in response to the microenvironment and include proteins, metabolites, RNA species, and nucleic acids. Novel findings on the biogenesis and fate of these vesicles reveal their fundamental role in cancer progression, with foreseeable and not-far-to-come clinical applications in NSCLC.