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.

Human erythroid progenitors express antigen presentation machinery.

Early-life immune exposures can profoundly impact lifelong health. However, functional mechanisms underlying fetal immune development remain incomplete. Erythrocytes are not typically considered active immune mediators, primarily because erythroid precursors discard their organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Erythroid progenitors and precursors circulate in human fetuses and neonates. Although there is limited evidence that erythroid precursors are immunomodulatory, our understanding of the underlying mechanisms remains inadequate. To define the immunobiological role of fetal and perinatal erythroid progenitors and precursors, we analyzed single cell RNA-sequencing data and found that transcriptomics support erythroid progenitors as putative immune mediators. Unexpectedly, we discovered that human erythroid progenitors constitutively express Major Histocompatibility Complex (MHC) class II antigen processing and presentation machinery, which are hallmarks of specialized antigen presenting immune cells. Furthermore, we demonstrate that erythroid progenitors internalize and cleave foreign proteins into peptide antigens. Unlike conventional antigen presenting cells, erythroid progenitors express atypical costimulatory molecules and immunoregulatory cytokines that direct the development of regulatory T cells, which are critical for establishing maternal-fetal tolerance. Expression of MHC II in definitive erythroid progenitors begins during the second trimester, coinciding with the appearance of mature T cells in the fetus, and is absent in primitive progenitors. Lastly, we demonstrate physical and molecular interaction potential of erythroid progenitors and T cells in the fetal liver. Our findings shed light on a unique orchestrator of fetal immunity and provide insight into the mechanisms by which erythroid cells contribute to host defense.

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.

Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice.

Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibody to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.

Extracellular domains of CARs reprogramme T cell metabolism without antigen stimulation.

Metabolism is an indispensable part of T cell proliferation, activation and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are composed of extracellular domains-often single-chain variable fragments (scFvs)-that determine ligand specificity and intracellular domains that trigger signalling following antigen binding. Here, we show that CARs differing only in the scFv variously reprogramme T cell metabolism. Even without exposure to antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observed basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harbouring a rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14G2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Modest overflow metabolism of CAR-T cells and metabolic compatibility between cancer cells and CAR-T cells are identified as features of efficacious CAR-T cell therapy.

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.

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer.

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

c-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity.

During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.

Decision-Making During High-Risk Events: A Systematic Literature Review.

Effective decision-making in crisis events is challenging due to time pressure, uncertainty, and dynamic decisional environments. We conducted a systematic literature review in PubMed and PsycINFO, identifying 32 empiric research papers that examine how trained professionals make naturalistic decisions under pressure. We used structured qualitative analysis methods to extract key themes. The studies explored different aspects of decision-making across multiple domains. The majority (19) focused on healthcare; military, fire and rescue, oil installation, and aviation domains were also represented. We found appreciable variability in research focus, methodology, and decision-making descriptions. We identified five main themes: (1) decision-making strategy, (2) time pressure, (3) stress, (4) uncertainty, and (5) errors. Recognition-primed decision-making (RPD) strategies were reported in all studies that analyzed this aspect. Analytical strategies were also prominent, appearing more frequently in contexts with less time pressure and explicit training to generate multiple explanations. Practitioner experience, time pressure, stress, and uncertainty were major influencing factors. Professionals must adapt to the time available, types of uncertainty, and individual skills when making decisions in high-risk situations. Improved understanding of these decisional factors can inform evidence-based enhancements to training, technology, and process design.

Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response.

The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.

Freestanding Ambulatory Surgery Centers and Patients Undergoing Outpatient Knee Arthroplasty.

Importance: In 2018, Medicare removed total knee arthroplasty from the list of inpatient-only procedures, resulting in a new pool of patients eligible for outpatient total knee arthroplasty. How this change was associated with the characteristics of patients undergoing outpatient knee arthroplasty at hospital-owned surgery centers (HOSCs) vs freestanding ambulatory surgery centers (FASCs) is unknown. Objectives: To describe the characteristics of patients undergoing outpatient, elective total and partial knee arthroplasty in 2017 and 2018 and to compare the cohorts receiving treatment at FASCs and HOSCs. Design, Setting, and Participants: This observational retrospective cohort study included 5657 patients having elective, outpatient partial and total knee arthroplasty in the Florida and Wisconsin State Ambulatory Surgery Databases in 2017 and 2018. Prior admissions were identified in the State Inpatient Database. Statistical analysis was performed from March to June 2022. Main Outcomes and Measures: Characteristics of patients undergoing surgery at a FASC vs a HOSC in 2017 and 2018 were compared. Results: A total of 5657 patients (mean [SD] age, 64.2 [9.9] years; 2907 women [51.4%]) were included in the study. Outpatient knee arthroplasties increased from 1910 in 2017 to 3747 in 2018 and were associated with an increase in total knee arthroplasties (474 in 2017 vs 2065 in 2018). The influx of patients undergoing outpatient knee arthroplasty was associated with an amplification of differences between the patients treated at FASCs and the patients treated at HOSCs. Patients with private payer insurance seen at FASCs increased from 63.4% in 2017 (550 of 867) to 72.7% in 2018 (1272 of 1749) (P < .001), while the percentage of patients with private payer insurance seen at HOSCs increased, but to a lesser extent (41.6% [427 of 1027] in 2017 vs 46.4% [625 of 1346] in 2018; P < .001). In 2017, the percentages of White patients seen at FASCs and HOSCs were similar (85.0% [737 of 867] vs 88.2% [906 of 1027], respectively); in 2018, the percentage of White patients seen at FASCs had increased and was significantly different from the percentage of White patients seen at HOSCs (90.6% [1585 of 1749] vs 87.9% [1183 of 1346]; P = .01). Both types of facilities saw an increase from 2017 to 2018 in the percentage of patients from communities of low social vulnerability, but this increase was greater for FASCs (FASCs: 6.7% [58 of 867] in 2017 vs 33.9% [593 of 1749] in 2018; HOSCs: 7.6% [78 of 1027] in 2017 vs 21.2% [285 of 1346] in 2018). Finally, while FASCs and HOSCs had cared for a similar portion of patients with prior admissions in 2017 (7.8% [68 of 867] vs 9.4% [97 of 1027], respectively; P = .25), in 2018, FASCs cared for fewer patients with prior admissions than HOSCs (4.0% [70 of 1749] vs 8.1% [109 of 1346]; P < .001). Conclusions: This study suggests that the increase in the number of patients undergoing outpatient knee arthroplasty in 2018 corresponded to FASCs treating a greater share of patients who were White, covered by private payer insurance, and healthier. These findings raise a concern that as more operations transition to the outpatient setting, variability in access to FASCs may increase, leaving hospital-owned centers to bear a greater share of the burden of caring for more vulnerable patients with more severe illness.

Association between "Balance Billing" Legislation and Anesthesia Payments in California: A Retrospective Analysis.

BACKGROUND: Insured patients who receive out-of-network care may receive a "balance bill" for the difference between the practitioner's charge and their insurer's contracted rate. In 2017, California banned balance billing for anesthesia care. This study examined the association between California's law and subsequent payments for anesthesia care. The authors hypothesized that, after the law's implementation, there would be no change in in-network payment amounts, and that out-of-network payment amounts and the portion of claims occurring out-of-network would decline. METHODS: The study used average, quarterly, California county-level payment data (2013 to 2020) derived from a claims database of commercially insured patients. Using a difference-in-differences approach, the change was estimated in payment amounts for intraoperative or intrapartum anesthesia care, along with the portion of claims occurring out-of-network, after the law's implementation. The comparison group was office visit payments, expected to be unaffected by the law. The authors prespecified that they would refer to differences of 10% or greater as policy significant. RESULTS: The sample consisted of 43,728 procedure code-county-quarter-network combinations aggregated from 4,599,936 claims. The law's implementation was associated with a significant 13.6% decline in payments for out-of-network anesthesia care (95% CI, -16.5 to -10.6%; P < 0.001), translating to an average $108 decrease across all procedures (95% CI, -$149 to -$64). There was a statistically significant 3.0% increase in payments for in-network anesthesia care (95% CI, 0.9 to 5.1%; P = 0.007), translating to an average $87 increase (95% CI, $64 to $110), which may be notable in some circumstances but did not meet the study threshold for identifying a change as policy significant. There was a nonstatistically significant increase in the portion of claims occurring out-of-network (10.0%, 95% CI, -4.1 to 24.2%; P = 0.155). CONCLUSIONS: California's balance billing law was associated with significant declines in out-of-network anesthesia payments in the first 3 yr after implementation. There were mixed statistical and policy significant results for in-network payments and the proportion of out-of-network claims.

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.

High Voltage: The Molecular Properties of Redox-Active Dissolved Organic Matter in Northern High-Latitude Lakes.

Redox-active functional groups in dissolved organic matter (DOM) are crucial for microbial electron transfer and methane emissions. However, the extent of aquatic DOM redox properties across northern high-latitude lakes and their relationships with DOM composition have not been thoroughly described. We quantified electron donating capacity (EDC) and electron accepting capacity (EAC) in lake DOM from Canada to Alaska and assessed their relationships with parameters from absorbance, fluorescence, and ultrahigh resolution mass spectrometry (FT-ICR MS) analyses. EDC and EAC are strongly tied to aromaticity and negatively related to aliphaticity and protein-like content. Redox-active formulae spanned a range of aromaticity, including highly unsaturated phenolic formulae, and correlated negatively with many aliphatic N and S-containing formulae. This distribution illustrates the compositional diversity of redox-sensitive functional groups and their sensitivity to ecosystem properties such as local hydrology and residence time. Finally, we developed a reducing index (RI) to predict EDC in aquatic DOM from FT-ICR MS spectra and assessed its robustness using riverine DOM. As the hydrology of the northern high-latitudes continues to change, we expect differences in the quantity and partitioning of EDC and EAC within these lakes, which have implications for local water quality and methane emissions.

Extracellular Domains of CAR Reprogram T-Cell Metabolism Without Antigen Stimulation.

Metabolism is an indispensable part of T-cell proliferation, activation, and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are comprised of extracellular domains that determine cancer specificity, often using single-chain variable fragments (scFvs), and intracellular domains that trigger signaling upon antigen binding. Here we show that CARs differing only in the scFv reprogram T-cell metabolism differently. Even in the absence of antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observe basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harboring rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14g2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Since CAR-dependent metabolic reprogramming alters cellular energetics, nutrient utilization, and proliferation, metabolic profiling should be an integral part of CAR-T cell development.

Dyes for guest-host liquid crystal applications: a general approach to the rapid computational assessment of useful molecular designs.

A set of experimental dichroic order parameters ranging from ca. +0.66 to -0.22 was obtained by recording polarized UV-visible absorption spectra from aligned samples of fifteen different guest anthraquinone and azo dyes in the nematic host 4-cyano-4'-pentylbiphenyl (5CB). DFT-optimised structures were calculated for between 1 and 16 conformers/tautomers of each dye, and their relative energies, UV-visible absorption wavelengths, oscillator strengths, transition dipole moments, molecular surface tensors and quadrupole tensors were obtained and used in subsequent calculations. A simple approach provided calculated UV-visible absorption spectra of the dyes that gave a qualitative match with the experimental spectra, and the calculated peak positions showed a linear correlation with the experimental values across the full visible range of ca. 350-700 nm. A short-range, shape-based, mean-field orienting potential based on the calculated surface tensors was combined with the calculated transition dipole moment vectors to give calculated dichroic ratios of the dyes that showed a linear correlation across the full range of experimental values. A modification of this mean-field orienting potential to include a long-range, electrostatic component based on the calculated quadrupole tensors gave a slightly improved linear correlation but a slightly worse overall match to the experimental values. The results show that short-range, shape-based interactions dominate the orienting potential for the systems studied here, with the inclusion of long-range quadrupole interactions providing a slightly better model for only some of the dyes. Overall, the use of a mean-field approach allied with molecular properties that can be calculated with relative ease and low computational expense has provided calculated peak positions and dichroic ratios that show good matches and correlations with experimental data from a variety of dye structures without the need to input any experimental data from the dyes. Hence, this method may provide a general and rapid approach to predicting the optical properties of dyes in liquid crystal hosts, enabling candidate dye structures to be screened prior to synthesis.