Insights into the Clinical, Biological and Therapeutic Impact of Copy Number Alteration in Cancer.

Copy number alterations (CNAs), resulting from the gain or loss of genetic material from as little as 50 base pairs or as big as entire chromosome(s), have been associated with many congenital diseases, de novo syndromes and cancer. It is established that CNAs disturb the dosage of genomic regions including enhancers/promoters, long non-coding RNA and gene(s) among others, ultimately leading to an altered balance of key cellular functions. In cancer, CNAs have been associated with almost all steps of the disease: predisposition, initiation, development, maintenance, response to treatment, resistance, and relapse. Therefore, understanding how specific CNAs contribute to tumourigenesis may provide prognostic insight and ultimately lead to the development of new therapeutic approaches to improve patient outcomes. In this review, we provide a snapshot of what is currently known about CNAs and cancer, incorporating topics regarding their detection, clinical impact, origin, and nature, and discuss the integration of innovative genetic engineering strategies, to highlight the potential for targeting CNAs using novel, dosage-sensitive and less toxic therapies for CNA-driven cancer.

Precision-guided treatment in high-risk pediatric cancers.

Recent research showed that precision medicine can identify new treatment strategies for patients with childhood cancers. However, it is unclear which patients will benefit most from precision-guided treatment (PGT). Here we report consecutive data from 384 patients with high-risk pediatric cancer (with an expected cure rate of less than 30%) who had at least 18 months of follow-up on the ZERO Childhood Cancer Precision Medicine Program PRecISion Medicine for Children with Cancer (PRISM) trial. A total of 256 (67%) patients received PGT recommendations and 110 (29%) received a recommended treatment. PGT resulted in a 36% objective response rate and improved 2-year progression-free survival compared with standard of care (26% versus 12%; P = 0.049) or targeted agents not guided by molecular findings (26% versus 5.2%; P = 0.003). PGT based on tier 1 evidence, PGT targeting fusions or commenced before disease progression had the greatest clinical benefit. Our data show that PGT informed by comprehensive molecular profiling significantly improves outcomes for children with high-risk cancers. ClinicalTrials.gov registration: NCT03336931.

Biologic and clinical analysis of childhood gamma delta T-ALL identifies LMO2/STAG2 rearrangements as extremely high-risk.

Acute lymphoblastic leukemia expressing the gamma delta T cell receptor (yo T-ALL) is a poorly understood disease. We studied 200 children with yo T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. yo T-ALL diagnosed in children under three years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by Poly(ADP-ribose) polymerase (PARP) inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric yo T-ALL.

Minimising Adverse Drug Reactions and Verifying Economic Legitimacy-Pharmacogenomics Implementation in Children (MARVEL- PIC): protocol for a national randomised controlled trial of pharmacogenomics implementation.

INTRODUCTION: DNA-informed prescribing (termed pharmacogenomics, PGx) is the epitome of personalised medicine. Despite international guidelines existing, its implementation in paediatric oncology remains sparse. METHODS AND ANALYSIS: Minimising Adverse Drug Reactions and Verifying Economic Legitimacy-Pharmacogenomics Implementation in Children is a national prospective, multicentre, randomised controlled trial assessing the impact of pre-emptive PGx testing for actionable PGx variants on adverse drug reaction (ADR) incidence in patients with a new cancer diagnosis or proceeding to haematopoetic stem cell transplant. All ADRs will be prospectively collected by surveys completed by parents/patients using the National Cancer Institute Pediatric Patient Reported [Ped-PRO]-Common Terminology Criteria for Adverse Events (CTCAE) (weeks 1, 6 and 12). Pharmacist will assess for causality and severity in semistructured interviews using the CTCAE and Liverpool Causality Assessment Tool. The primary outcome is a reduction in ADRs among patients with actionable PGx variants, where an ADR will be considered as any CTCAE grade 2 and above for non-haematological toxicities and any CTCAE grade 3 and above for haematological toxicities Cost-effectiveness of pre-emptive PGx (secondary outcome) will be compared with standard of care using hospital inpatient and outpatient data along with the validated Childhood Health Utility 9D Instrument. Power and statistics considerations: A sample size of 440 patients (220 per arm) will provide 80% power to detect a 24% relative risk reduction in the primary endpoint of ADRs (two-sided α=5%, 80% vs 61%), allowing for 10% drop-out. ETHICS AND DISSEMINATION: The ethics approval of the trial has been obtained from the Royal Children's Hospital Ethics Committee (HREC/89083/RCHM-2022). The ethics committee of each participating centres nationally has undertaken an assessment of the protocol and governance submission. TRIAL REGISTRATION NUMBER: NCT05667766.

Murine bone-derived mesenchymal stem cells undergo molecular changes after a single passage in culture.

The rarity of the mesenchymal stem cell (MSC) population poses a significant challenge for MSC research. Therefore, these cells are often expanded in vitro, prior to use. However, long-term culture has been shown to alter primary MSC properties. Additionally, early passage primary MSCs in culture are often assumed to represent the primary MSC population in situ, however, little research has been done to support this. Here, we compared the transcriptomic profiles of murine MSCs freshly isolated from the bone marrow to those that had been expanded in culture for 10 days. We identified that a single passage in culture extensively altered MSC molecular signatures associated with cell cycling, differentiation and immune response. These findings indicate the critical importance of the MSC source, highlighting the need for optimization of culture conditions to minimize the impact on MSC biology and a transition towards in vivo methodologies for the study of MSC function.

Invasive fungal disease in children with solid tumors: An Australian multicenter 10-year review.

Invasive fungal disease (IFD) occurs less frequently during treatment for solid compared to hematological malignancies in children, and risk groups are poorly defined. Retrospective national multicenter cohort data (2004-2013) were analyzed to document prevalence, clinical characteristics, and microbiology of IFD. Amongst 2067 children treated for solid malignancy, IFD prevalence was 1.9% overall and 1.4% for proven/probable IFD. Of all IFD episodes, 42.5% occurred in patients with neuroblastoma (prevalence 7.0%). Candida species comprised 54.8% of implicated pathogens in proven/probable IFD. In children with solid tumors, IFD is rare, and predominantly caused by yeasts.Routine prophylaxis may not be warranted.

Reading and Writing the Ubiquitin Code Using Genetic Code Expansion.

Deciphering ubiquitin proteoform signaling and its role in disease has been a long-standing challenge in the field. The effects of ubiquitin modifications, its relation to ubiquitin-related machineries, and its signaling output has been particularly limited by its reconstitution and means of characterization. Advances in genetic code expansion have contributed towards addressing these challenges by precision incorporation of unnatural amino acids through site selective codon suppression. This review discusses recent advances in studying the 'writers', 'readers', and 'erasers' of the ubiquitin code using genetic code expansion. Highlighting strategies towards genetically encoded protein ubiquitination, ubiquitin phosphorylation, acylation, and finally surveying ubiquitin interactions, we strive to bring attention to this unique approach towards addressing a widespread proteoform problem.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML.

BACKGROUND: Hematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due to relapse. To improve overall survival, we propose a combined strategy based on cord blood (CB)-HCT with the application of AML-specific T cell receptor (TCR)-engineered T cell therapy derived from the same CB graft. METHODS: We produced CB-CD8+ T cells expressing a recombinant TCR (rTCR) against Wilms tumor 1 (WT1) while lacking endogenous TCR (eTCR) expression to avoid mispairing and competition. CRISPR-Cas9 multiplexing was used to target the constant region of the endogenous TCRα (TRAC) and TCRß (TRBC) chains. Next, an optimized method for lentiviral transduction was used to introduce recombinant WT1-TCR. The cytotoxic and migration capacity of the product was evaluated in coculture assays for both cell lines and primary pediatric AML blasts. RESULTS: The gene editing and transduction procedures achieved high efficiency, with up to 95% of cells lacking eTCR and over 70% of T cells expressing rWT1-TCR. WT1-TCR-engineered T cells lacking the expression of their eTCR (eTCR-/- WT1-TCR) showed increased cell surface expression of the rTCR and production of cytotoxic cytokines, such as granzyme A and B, perforin, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), on antigen recognition when compared with WT1-TCR-engineered T cells still expressing their eTCR (eTCR+/+ WT1-TCR). CRISPR-Cas9 editing did not affect immunophenotypic characteristics or T cell activation and did not induce increased expression of inhibitory molecules. eTCR-/- WT1-TCR CD8+ CB-T cells showed effective migratory and killing capacity in cocultures with neoplastic cell lines and primary AML blasts, but did not show toxicity toward healthy cells. CONCLUSIONS: In summary, we show the feasibility of developing a potent CB-derived CD8+ T cell product targeting WT1, providing an option for post-transplant allogeneic immune cell therapy or as an off-the-shelf product, to prevent relapse and improve the clinical outcome of children with AML.

Pharmacokinetics of PEGasparaginase in Infants with Acute Lymphoblastic Leukemia.

BACKGROUND: PEGasparaginase is known to be a critical drug for treating pediatric acute lymphoblastic leukemia (ALL), however, there is insufficient evidence to determine the optimal dose for infants who are less than one year of age at diagnosis. This international study was conducted to identify the pharmacokinetics of PEGasparaginase in infants with newly diagnosed ALL and gather insight into the clearance and dosing of this population. METHODS: Infants with ALL who received treatment with PEGasparaginase were included in our population pharmacokinetic assessment employing non-linear mixed effects modelling (NONMEM). RESULTS: 68 infants with ALL, with a total of 388 asparaginase activity samples, were included. PEGasparaginase doses ranging from 400 to 3,663 IU/m2 were administered either intravenously or intramuscularly. A one-compartment model with time-dependent clearance, modeled using a transit model, provided the best fit to the data. Body weight was significantly correlated with clearance and volume of distribution. The final model estimated a half-life of 11.7 days just after administration, which decreased to 1.8 days 14 days after administration. Clearance was 19.5% lower during the post-induction treatment phase compared to induction. CONCLUSION: The pharmacokinetics of PEGasparaginase in infants diagnosed under one year of age with ALL is comparable to that of older children (1-18 years). We recommend a PEGasparaginase dosing at 1,500 IU/m2 for infants without dose adaptations according to age, and implementing therapeutic drug monitoring as standard practice.

The critical role of the bone marrow stromal microenvironment for the development of drug screening platforms in leukemia.

Extensive research over the past 50 years has resulted in significant improvements in survival for patients diagnosed with leukemia. Despite this, a subgroup of patients harboring high-risk genetic alterations still suffer from poor outcomes. There is a desperate need for new treatments to improve survival, yet consistent failure exists in the translation of in vitro drug development to clinical application. Preclinical screening conventionally utilizes tumor cell monocultures to assess drug activity; however, emerging research has acknowledged the vital role of the tumor microenvironment in treatment resistance and disease relapse. Current co-culture drug screening methods frequently employ fibroblasts as the designated stromal cell component. Alternative stromal cell types that are known to contribute to chemoresistance are often absent in preclinical evaluations of drug efficacy. This review highlights mechanisms of chemoresistance by a range of different stromal constituents present in the bone marrow microenvironment. Utilizing an array of stromal cell types at the early stages of drug screening may enhance the translation of in vitro drug development to clinical use. Ultimately, we highlight the need to consider the bone marrow microenvironment in drug screening platforms for leukemia to develop superior therapies for the treatment of high-risk patients with poor prognostic outcomes.

FDA-approved disulfiram as a novel treatment for aggressive leukemia.

Acute leukemia continues to be a major cause of death from disease worldwide and current chemotherapeutic agents are associated with significant morbidity in survivors. While better and safer treatments for acute leukemia are urgently needed, standard drug development pipelines are lengthy and drug repurposing therefore provides a promising approach. Our previous evaluation of FDA-approved drugs for their antileukemic activity identified disulfiram, used for the treatment of alcoholism, as a candidate hit compound. This study assessed the biological effects of disulfiram on leukemia cells and evaluated its potential as a treatment strategy. We found that disulfiram inhibits the viability of a diverse panel of acute lymphoblastic and myeloid leukemia cell lines (n = 16) and patient-derived xenograft cells from patients with poor outcome and treatment-resistant disease (n = 15). The drug induced oxidative stress and apoptosis in leukemia cells within hours of treatment and was able to potentiate the effects of daunorubicin, etoposide, topotecan, cytarabine, and mitoxantrone chemotherapy. Upon combining disulfiram with auranofin, a drug approved for the treatment of rheumatoid arthritis that was previously shown to exert antileukemic effects, strong and consistent synergy was observed across a diverse panel of acute leukemia cell lines, the mechanism of which was based on enhanced ROS induction. Acute leukemia cells were more sensitive to the cytotoxic activity of disulfiram than solid cancer cell lines and non-malignant cells. While disulfiram is currently under investigation in clinical trials for solid cancers, this study provides evidence for the potential of disulfiram for acute leukemia treatment. KEY MESSAGES: Disulfiram induces rapid apoptosis in leukemia cells by boosting oxidative stress. Disulfiram inhibits leukemia cell growth more potently than solid cancer cell growth. Disulfiram can enhance the antileukemic efficacy of chemotherapies. Disulfiram strongly synergises with auranofin in killing acute leukemia cells by ROS induction. We propose testing of disulfiram in clinical trial for patients with acute leukemia.

Biocatalytic cyclization of small macrolactams by a penicillin-binding protein-type thioesterase.

Macrocyclic peptides represent promising scaffolds for chemical tools and potential therapeutics. Synthetic methods for peptide macrocyclization are often hampered by C-terminal epimerization and oligomerization, leading to difficult scalability. While chemical strategies to circumvent this issue exist, they often require specific amino acids to be present in the peptide sequence. Herein, we report the characterization of Ulm16, a peptide cyclase belonging to the penicillin-binding protein-type class of thioesterases that catalyze head-to-tail macrolactamization of nonribosmal peptides. Ulm16 efficiently cyclizes various nonnative peptides ranging from 4 to 6 amino acids with catalytic efficiencies of up to 3 × 106 M-1 s-1. Unlike many previously described homologs, Ulm16 tolerates a variety of C- and N-terminal amino acids. The crystal structure of Ulm16, along with modeling of its substrates and site-directed mutagenesis, allows for rationalization of this wide substrate scope. Overall, Ulm16 represents a promising tool for the biocatalytic production of macrocyclic peptides.

Reproducible Bioinformatics Analysis Workflows for Detecting IGH Gene Fusions in B-Cell Acute Lymphoblastic Leukaemia Patients.

B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by diverse genomic alterations, the most frequent being gene fusions detected via transcriptomic analysis (mRNA-seq). Due to its hypervariable nature, gene fusions involving the Immunoglobulin Heavy Chain (IGH) locus can be difficult to detect with standard gene fusion calling algorithms and significant computational resources and analysis times are required. We aimed to optimize a gene fusion calling workflow to achieve best-case sensitivity for IGH gene fusion detection. Using Nextflow, we developed a simplified workflow containing the algorithms FusionCatcher, Arriba, and STAR-Fusion. We analysed samples from 35 patients harbouring IGH fusions (IGH::CRLF2 n = 17, IGH::DUX4 n = 15, IGH::EPOR n = 3) and assessed the detection rates for each caller, before optimizing the parameters to enhance sensitivity for IGH fusions. Initial results showed that FusionCatcher and Arriba outperformed STAR-Fusion (85-89% vs. 29% of IGH fusions reported). We found that extensive filtering in STAR-Fusion hindered IGH reporting. By adjusting specific filtering steps (e.g., read support, fusion fragments per million total reads), we achieved a 94% reporting rate for IGH fusions with STAR-Fusion. This analysis highlights the importance of filtering optimization for IGH gene fusion events, offering alternative workflows for difficult-to-detect high-risk B-ALL subtypes.

Editorial: Optimizing opioid prescriptions in the emergency department.

Optimizing opioid prescriptions in the emergency department is essential to address the opioid pandemic while ensuring patient wellbeing. This requires a comprehensive approach that includes exploring alternatives to opioids for pain management, identifying individuals at risk for opioid addiction, implementing evidence-based guidelines, and involving doctors in the management of opioid addiction.

Alzheimer's disease: a mini-review for the clinician.

Alzheimer's disease (AD), the most common form of dementia, is a striking example of the connection between neurophysiological abnormalities and higher-order cognitive deficiencies. Since its initial description in 1906, research into the pathophysiology and etiology of AD has led to the illumination of an incredibly complex set of genetic and molecular mechanisms for the disease's progression, characterized by much more than the neuropathological hallmarks of beta-amyloid (Aß) plaques and neurofibrillary tangles (NFTs). In this review, findings relating the neurodegeneration present in AD to its clinical presentation and treatment are summarized, with an emphasis on the interconnectedness of disease pathophysiology. Further, diagnostic guidelines are provided based on the National Institute on Aging-Alzheimer's Association (NIA-AA) workgroup's clinical recommendations. Through the dissemination of detailed but digestible open access resources such as this one, we can move towards an increase in the equity and accessibility of education for the modern clinician.

Acquisition and annotation in high resolution in vivo digital biopsy by confocal microscopy for diagnosis in oral precancer and cancer.

Introduction: Scanned fibre endomicroscopes are full point-scanning confocal microscopes with submicron lateral resolution with an optical slice thickness thin enough to isolate individual cell layers, allow active positioning of the optical slice in the z-axis and collection of megapixel images. Here we present descriptive findings and a brief atlas of an acquisition and annotation protocol high resolution in vivo capture of oral mucosal pathology including oral squamous cell carcinoma and dysplasia using a fluorescence scanned fibre endomicroscope with 3 topical fluorescent imaging agents: fluorescein, acriflavine and PARPi-FL. Methods: Digital biopsy was successfully performed via an acquisition protocol in seventy-one patients presenting for investigation of oral mucosal abnormalities using a miniaturized, handheld scanned fibre endoscope. Multiple imaging agents were utilized and multiple time points sampled. Fifty-nine patients had a matched histopathology correlating in location with imaging. The images were annotated back to macrographic location using a purpose-built software, MouthMap™. Results: Acquisition and annotation of cellular level resolved images was demonstrated with all 3 topical agents. Descriptive observations between clinically or histologically normal oral mucosa showed regular intranuclear distance, a regular nuclear profile and fluorescent homogeneity. This was dependent on the intraoral location and type of epithelium being observed. Key features of malignancy were a loss of intranuclear distance, disordered nuclear clustering and irregular nuclear fluorescence intensity and size. Perinuclear fluorescent granules were seen in the absence of irregular nuclear features in lichenoid inflammation. Discussion: High resolution oral biopsy allows for painless and rapid capture of multiple mucosal sites, resulting in more data points to increase diagnostic precision. High resolution digital micrographs can be easily compared serially across multiple time points utilizing an annotation software. In the present study we have demonstrated realization of a high-resolution digital biopsy protocol of the oral mucosa for utility in the diagnosis of oral cancer and precancer..

Delivery of PEGylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia.

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia.