Circulating tumor cells: advancing personalized therapy in small cell lung cancer patients.

Small cell lung cancer (SCLC) is a highly aggressive cancer with a dismal 5-year survival of < 7%, despite the addition of immunotherapy to first-line chemotherapy. Specific tumor biomarkers, such as delta-like ligand 3 (DLL3) and schlafen11 (SLFN11), may enable the selection of more efficacious, novel immunomodulating targeted treatments like bispecific T-cell engaging monoclonal antibodies (tarlatamab) and chemotherapy with PARP inhibitors. However, obtaining a tissue biopsy sample can be challenging in SCLC. Circulating tumor cells (CTCs) have the potential to provide molecular insights into a patient's cancer through a "simple" blood test. CTCs have been studied for their prognostic ability in SCLC; however, their value in guiding treatment decisions is yet to be elucidated. This review explores novel and promising targeted therapies in SCLC, summarizes current knowledge of CTCs in SCLC, and discusses how CTCs can be utilized for precision medicine.

Circulating tumor cell detection may offer earlier diagnosis in patients suspected of asbestos-related lung cancer.

Asbestos-Related Lung Cancer (ARLC) presents ongoing diagnostic challenges despite improved imaging technologies. The long latency period, coupled with limited access to occupational and environmental data along with the confounding effects of smoking and other carcinogens adds complexity to the diagnostic process. Compounding these challenges is the absence of a specific histopathologic or mutational signature of ARLC. A correlation between PD-L1 expression and response to immune checkpoint inhibition has not yet been proven. Thus, new biomarkers are needed to allow accurate diagnoses of ARLC, to enable prognostication and to offer personalized treatments. Liquid biopsies, encompassing circulating DNA and circulating tumor cells (CTCs), have gained attention as novel diagnostic methods in lung cancer to screen high-risk populations including those exposed to asbestos. CTCs can be enumerated and molecularly profiled to provide predictive and prognostic information. CTC studies have not been undertaken in populations at risk of ARLC to date. The potential of CTCs to provide real-time molecular insight into ARLC biology may significantly improve the diagnosis and management of ARLC patients.

Two-year safety outcomes of iPS cell-derived mesenchymal stromal cells in acute steroid-resistant graft-versus-host disease.

The first completed clinical trial of induced pluripotent stem cell (iPS cell)-derived cells was conducted in 15 participants with steroid-resistant acute graft-versus-host disease. After intravenous infusion of mesenchymal stromal cells (CYP-001 derived from a clone of human iPS cells), we reported the safety, tolerability and efficacy within the primary evaluation period at day 100. We now report results at the 2-year follow-up: 9 of 15 (60%) participants survived, which compares favorably with previously reported outcomes in studies of steroid-resistant acute graft-versus-host disease. Causes of death were complications commonly observed in recipients of allogeneic hematopoietic stem cell transplantation, and not considered by the investigators to be related to CYP-001 treatment. There were no serious adverse events, tumors or other safety concerns related to CYP-001. In conclusion, systemic delivery of iPS cell-derived cells was safe and well tolerated over 2 years of follow-up, with sustained outcomes up to 2 years after the first infusion. ClinicalTrials.gov registration: NCT02923375 .

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

Mesothelin antigen density influences anti-mesothelin chimeric antigen receptor T cell cytotoxicity.

BACKGROUND AIMS: Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. METHODS: MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. RESULTS: SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. CONCLUSIONS: These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.

Understanding AAV vector immunogenicity: from particle to patient.

Gene therapy holds promise for patients with inherited monogenic disorders, cancer, and rare genetic diseases. Naturally occurring adeno-associated virus (AAV) offers a well-suited vehicle for clinical gene transfer due to its lack of significant clinical pathogenicity and amenability to be engineered to deliver therapeutic transgenes in a variety of cell types for long-term sustained expression. AAV has been bioengineered to produce recombinant AAV (rAAV) vectors for many gene therapies that are approved or in late-stage development. However, ongoing challenges hamper wider use of rAAV vector-mediated therapies. These include immunity against rAAV vectors, limited transgene packaging capacity, sub-optimal tissue transduction, potential risks of insertional mutagenesis and vector shedding. This review focuses on aspects of immunity against rAAV, mediated by anti-AAV neutralizing antibodies (NAbs) arising after natural exposure to AAVs or after rAAV vector administration. We provide an in-depth analysis of factors determining AAV seroprevalence and examine clinical approaches to managing anti-AAV NAbs pre- and post-vector administration. Methodologies used to quantify anti-AAV NAb levels and strategies to overcome pre-existing AAV immunity are also discussed. The broad adoption of rAAV vector-mediated gene therapies will require wider clinical appreciation of their current limitations and further research to mitigate their impact.

SpliceWiz: interactive analysis and visualization of alternative splicing in R.

Alternative splicing (AS) is a crucial mechanism for regulating gene expression and isoform diversity in eukaryotes. However, the analysis and visualization of AS events from RNA sequencing data remains challenging. Most tools require a certain level of computer literacy and the available means of visualizing AS events, such as coverage and sashimi plots, have limitations and can be misleading. To address these issues, we present SpliceWiz, an R package with an interactive Shiny interface that allows easy and efficient AS analysis and visualization at scale. A novel normalization algorithm is implemented to aggregate splicing levels within sample groups, thereby allowing group differences in splicing levels to be accurately visualized. The tool also offers downstream gene ontology enrichment analysis, highlighting ASEs belonging to functional pathways of interest. SpliceWiz is optimized for speed and efficiency and introduces a new file format for coverage data storage that is more efficient than BigWig. Alignment files are processed orders of magnitude faster than other R-based AS analysis tools and on par with command-line tools. Overall, SpliceWiz streamlines AS analysis, enabling reliable identification of functionally relevant AS events for further characterization. SpliceWiz is a Bioconductor package and is also available on GitHub (https://github.com/alexchwong/SpliceWiz).

The 4-1BBζ costimulatory domain in chimeric antigen receptors enhances CD8+ T-cell functionality following T-cell receptor stimulation.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of CD19- and B-cell maturation antigen-positive haematological malignancies. However, the effect of a CAR construct on the function of T-cells stimulated via their endogenous T-cell receptors (TCRs) has yet to be comprehensively investigated. METHODS: Experiments were performed to systematically assess TCR signalling and function in CAR T-cells using anti-mesothelin human CAR T-cells as a model system. CAR T-cells expressing the CD28 or 4-1BB costimulatory endodomains were manufactured and compared to both untransduced T-cells and CAR T-cells with a non-functional endodomain. These cell products were treated with staphylococcal enterotoxin B to stimulate the TCR, and in vitro functional assays were performed by flow cytometry. RESULTS: Increased proliferation, CD69 expression and IFNγ production were identified in CD8+ 4-1BBζ CAR T-cells compared to control untransduced CD8+ T-cells. These functional differences were associated with higher levels of phosphorylated ZAP70 after stimulation. In addition, these functional differences were associated with a differing immunophenotype, with a more than two-fold increase in central memory cells in CD8+ 4-1BBζ CAR T-cell products. CONCLUSION: Our data indicate that the 4-1BBζ CAR enhances CD8+ TCR-mediated function. This could be beneficial if the TCR targets epitopes on malignant tissues or infectious agents, but detrimental if the TCR targets autoantigens.

Evolutionary dynamics of whole-body regeneration across planarian flatworms.

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities.

International Society for Cell & Gene Therapy Position Paper: Key considerations to support evidence-based cell and gene therapies and oppose marketing of unproven products.

The field of regenerative medicine, including cellular immunotherapies, is on a remarkable growth trajectory. Dozens of cell-, tissue- and gene-based products have received marketing authorization worldwide while hundreds-to-thousands are either in preclinical development or under clinical investigation in phased clinical trials. However, the promise of regenerative therapies has also given rise to a global industry of direct-to-consumer offerings of prematurely commercialized cell and cell-based products with unknown safety and efficacy profiles. Since its inception, the International Society for Cell & Gene Therapy Committee on the Ethics of Cell and Gene Therapy has opposed the premature commercialization of unproven cell- and gene-based interventions and supported the development of evidence-based advanced therapy products. In the present Guide, targeted at International Society for Cell & Gene Therapy members, we analyze this industry, focusing in particular on distinctive features of unproven cell and cell-based products and the use of tokens of scientific legitimacy as persuasive marketing devices. We also provide an overview of reporting mechanisms for patients who believe they have been harmed by administration of unapproved and unproven products and suggest practical strategies to address the direct-to-consumer marketing of such products. Development of this Guide epitomizes our continued support for the ethical and rigorous development of cell and cell-based products with patient safety and therapeutic benefit as guiding principles.

Too little, too soon: Japan's experiment in regenerative medicine deregulation.

In 2013, Japan established a conditional approval pathway for cell and gene therapies. Our analysis of the four products approved via this pathway identifies evidence suggesting shortcomings in safety and efficacy data submitted by the sponsors and raises concerns about whether this pathway is delivering on its promise.

Accurate isolation and detection of circulating tumor cells using enrichment-free multiparametric high resolution imaging.

Introduction: The reliable and accurate detection of rare circulating tumor cells (CTCs) from cancer patient blood samples promises advantages in both research and clinical applications. Numerous CTC detection methods have been explored that rely on either the physical properties of CTCs such as density, size, charge, and/or their antigen expression profiles. Multiple factors can influence CTC recovery including blood processing method and time to processing. This study aimed to examine the accuracy and sensitivity of an enrichment-free method of isolating leukocytes (AccuCyte® system) followed by immunofluorescence staining and high-resolution imaging (CyteFinder® instrument) to detect CTCs. Method: Healthy human blood samples, spiked with cancer cells from cancer cell lines, as well as blood samples obtained from 4 subjects diagnosed with cancer (2 pancreatic, 1 thyroid, and 1 small cell lung) were processed using the AccuCyte-CyteFinder system to assess recovery rate, accuracy, and reliability over a range of processing times. Results: The AccuCyte-CyteFinder system was highly accurate (95.0%) at identifying cancer cells in spiked-in samples (in 7.5 mL of blood), even at low spiked-in numbers of 5 cells with high sensitivity (90%). The AccuCyte-CyteFinder recovery rate (90.9%) was significantly higher compared to recovery rates obtained by density gradient centrifugation (20.0%) and red blood cell lysis (52.0%). Reliable and comparable recovery was observed in spiked-in samples and in clinical blood samples processed up to 72 hours post-collection. Reviewer analysis of images from spiked-in and clinical samples resulted in high concordance (R-squared value of 0.998 and 0.984 respectively). Discussion: The AccuCyte-CyteFinder system is as an accurate, sensitive, and clinically practical method to detect and enumerate cancer cells. This system addresses some of the practical logistical challenges in incorporating CTCs as part of routine clinical care. This could facilitate the clinical use of CTCs in guiding precision, personalized medicine.

High mesothelin expression by immunohistochemistry predicts improved survival in pleural mesothelioma.

AIMS: Mesothelin (MSLN) is a cancer-associated antigen that is overexpressed in malignancies such as mesothelioma, pancreatic and ovarian cancer. It is also a target for novel personalised therapies, including antibodies, antibody-drug conjugates and chimeric antigen receptor T cells. Immunohistochemistry may predict those who would best respond to anti-mesothelin therapies and guide decisions in therapeutic strategy. This study aimed to assess the intensity and distribution of MSLN immunostaining in mesothelioma, and to determine the prognostic value of MSLN expression by histochemical-score (H-score). METHODS AND RESULTS: The MN1 anti-MSLN antibody was used to stain a formalin-fixed paraffin-embedded tissue microarray of histologically confirmed mesothelioma from 75 consecutive patients who had undergone pleurectomy with or without decortication. MSLN positivity, the staining intensity, distribution of staining and H-score were evaluated. The correlation of H-score with prognosis was investigated. Sixty-six per cent of epithelioid tumours were MSLN-positive (with expression in > 5% tumour cells). Of MSLN-expressing epithelioid tumours, 70.4% had moderate (2+) or strong (3+) intensity MSLN immunostaining, although only 37% of samples had staining in ≥ 50% of tumour cells. In multivariate analysis, MSLN H-score as a continuous variable and an H-score ≥ 33 were independent predictors of improved survival (P = 0.04 and P < 0.001, respectively). CONCLUSIONS: MSLN expression was more heterogenous in epithelioid mesothelioma than reported previously. Therefore, it would be appropriate to perform an immunohistochemical assessment of MSLN expression to stratify and assess patient suitability for mesothelin-targeted personalised therapies, such as chimeric antigen receptor T cells.

SARS-CoV-2-neutralising antibody BGB-DXP593 in mild-to-moderate COVID-19: a multicentre, randomised, double-blind, phase 2 trial.

Background: BGB-DXP593, a neutralising monoclonal antibody against SARS-CoV-2, has demonstrated strong activity in reducing viral RNA copy number in SARS-CoV-2-infected animal models. We aimed to examine the efficacy and safety of BGB-DXP593 in ambulatory patients with mild-to-moderate COVID-19. Methods: This global, randomised, double-blind, phase 2 study (ClinicalTrials.govNCT04551898) screened patients from 20 sites in Australia, Brazil, Mexico, South Africa, and the USA from December 2, 2020, through January 25, 2021. Patients with a first-positive SARS-CoV-2 test (positive reverse transcription-polymerase chain reaction test or authorised antigen test) ≤3 days before screening and mild-to-moderate COVID-19 symptoms for ≤7 days before treatment were randomised 1:1:1:1 to receive a single intravenous infusion of BGB-DXP593 5, 15, or 30 mg/kg, or placebo. The primary endpoint was change from baseline to Day 8 in viral RNA copies/mL as measured in nasopharyngeal swabs. Secondary endpoints were hospitalisation rate due to worsening COVID-19 and treatment-emergent adverse events (TEAEs). A prespecified exploratory endpoint was change in viral RNA copy number in saliva. Findings: Relative to the natural rate of clearance as assessed in placebo-exposed patients (-3.12 log10 copies/mL), no significant differences in nasopharygneal viral RNA copy number changes were observed (-2.93 to -3.63 log10 copies/mL) by Day 8 in BGB-DXP593-treated patients. Reductions from baseline to Day 8 in saliva viral RNA copy number were larger with BGB-DXP593 5 mg/kg (-1.37 log10 copies/mL [90% confidence interval -2.14, -0.61]; nominal p = 0.003) and 15 mg/kg (-1.26 [-2.06, -0.46]; nominal p = 0.01) vs placebo, and differences favoring BGB-DXP593 were observed by Day 3, although not statistically significant; no difference from placebo was observed for BGB-DXP593 30 mg/kg (-0.71 [-1.45, 0.04]; nominal p = 0.12). Hospitalisation rate due to COVID-19 was numerically lower with BGB-DXP593 (pooled: 2/134 patients; 1.5%) vs placebo (2/47 patients; 4.3%), although not statistically significant. Incidence of TEAEs was similar across treatment groups. No TEAE led to treatment discontinuation. Five serious TEAEs occurred, all attributed to COVID-19 pneumonia. Interpretation: BGB-DXP593 was well tolerated. Although nasopharyngeal swab SARS-CoV-2 viral RNA copy number was not significantly decreased compared with placebo, viral RNA copy number was inconsistently reduced by Day 8 in saliva at some doses as low as 5 mg/kg. Funding: BeiGene, Ltd.

Recent advances in cancer fusion transcript detection.

Extensive investigation of gene fusions in cancer has led to the discovery of novel biomarkers and therapeutic targets. To date, most studies have neglected chromosomal rearrangement-independent fusion transcripts and complex fusion structures such as double or triple-hop fusions, and fusion-circRNAs. In this review, we untangle fusion-related terminology and propose a classification system involving both gene and transcript fusions. We highlight the importance of RNA-level fusions and how long-read sequencing approaches can improve detection and characterization. Moreover, we discuss novel bioinformatic tools to identify fusions in long-read sequencing data and strategies to experimentally validate and functionally characterize fusion transcripts.

Towards resolution of the intron retention paradox in breast cancer.

BACKGROUND: After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR, and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue. METHODS: In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR. RESULTS: Strikingly, we found that aberrantly decreasing IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA-binding protein expression and changes in tissue composition are among the causes of aberrantly decreasing IR in BrCa. CONCLUSIONS: Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.

Increased chromatin accessibility facilitates intron retention in specific cell differentiation states.

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.

Illuminating the dark protein-protein interactome.

In living systems, a complex network of protein-protein interactions (PPIs) underlies most biochemical events. The human protein-protein interactome has been surveyed using yeast two-hybrid (Y2H)- and mass spectrometry (MS)-based approaches such as affinity purification coupled to MS (AP-MS). Despite decades of systematic investigations and collaborative multi-disciplinary efforts, there is no "gold standard" for documenting PPIs. A surprisingly large fraction of the human interactome remains uncharted, which we refer to as the "dark interactome." In this review, we highlight the complexity of the human interactome and discuss the current status of the human reference interactome maps. We discuss why a large proportion of the human interactome has remained refractory to traditional approaches. We propose an experimental model that can enable the identification of the dark interactome in a cell-type-specific manner. We also propose a framework to implement when embarking on studies designed to rigorously identify and characterize protein interactions.

Approved gene therapies in Australia: coming to a store near you.

Gene therapy has been promising paradigm-shifting advances in medical science for over two decades. Broadly, it is defined as a human therapy in which an existing defective gene function is added to, replaced, edited or disrupted to achieve a clinical benefit, up to and including a potential lifelong cure. Although originally set out to treat monogenic disorders, gene therapy has since been utilised to treat neoplasia, cardiovascular and neurodegenerative diseases, as well as infections. The realisation of this therapy has been dependent on the achievement of fundamental milestones in medicine, from determining the human genome sequence to identifying effective vehicles for the gene of interest, ultimately facilitating gene delivery in humans. In this review, six approved gene and cell therapies available in Australia are described. Their efficacy, adverse effects, limitations and eligibility are discussed, as well as an overview of cost and future directions.