Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning.

Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.

Recombination in bdelloid rotifer genomes: asexuality, transfer and stress.

Bdelloid rotifers constitute a class of microscopic animals living in freshwater habitats worldwide. Several strange features of bdelloids have drawn attention: their ability to tolerate desiccation and other stresses, a lack of reported males across the clade despite centuries of study, and unusually high numbers of horizontally acquired, non-metazoan genes. Genome sequencing is transforming our understanding of their lifestyle and its consequences, while in turn providing wider insights about recombination and genome organisation in animals. Many questions remain, not least how to reconcile apparent genomic signatures of sex with the continued absence of reported males, why bdelloids have so many horizontally acquired genes, and how their remarkable ability to survive stress interacts with recombination and other genomic processes.

In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice.

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1-4. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.

Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer.

ABSTRACT: Gene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e-4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

Search-and-replace genome editing without double-strand breaks or donor DNA.

Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2-5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.

Proximity to cancer rehabilitation and exercise oncology by geography, race, and socioeconomic status.

BACKGROUND: Cancer rehabilitation and exercise oncology (CR/EO) have documented benefits for people living with and beyond cancer. The authors examined proximity to CR/EO programs across the United States with respect to population density, race and ethnicity, socioeconomic status, and cancer incidence and mortality rates. METHODS: This cross-sectional study was conducted in 2022-2023. Online searches were initiated to identify CR/EO programs. Geocoding was used to obtain latitudinal and longitudinal geospatial coordinates. Demographic data were abstracted from the 2020 5-year American Community Survey. Cancer incidence and mortality data were obtained from the Centers for Disease Control and Prevention. US 2013 Rural-Urban Continuum Code (RUCC) classification was used to define counties as either urban (RUCC 1-3) or rural (RUCC 4-9). Multivariable logistic regression was used to evaluate the association between being far from a program and census-tract level factors. RESULTS: In total, 2133 CR/EO programs were identified nationwide. The distance from a program increased with decreasing population density: rural tracts were 17.68 ± 0.24 miles farther from a program compared with urban tracts (p < .001). Program proximity decreased as the neighborhood deprivation index increased (p < .001). Exercise oncology programs were less common than cancer rehabilitation programs in tracts with a larger proportion of minority residents (p < .001). CONCLUSIONS: Prior research has documented that underrepresented populations have worse cancer-related symptoms and higher cancer mortality. Herein, the authors document their findings that these same populations are less likely to have proximity to CR/EO programs, which are associated with improved cancer-related symptoms and cancer mortality outcomes. To realize the positive outcomes from CR/EO programming, efforts must focus on supporting expanded programming and sustainable payment for these services.

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.

Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

Bidirectional transport of amino acids regulates mTOR and autophagy.

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

COVID-19 and silent hypoxemia in a minimal closed-loop model of the respiratory rhythm generator.

Silent hypoxemia, or "happy hypoxia," is a puzzling phenomenon in which patients who have contracted COVID-19 exhibit very low oxygen saturation ( SaO 2 < 80%) but do not experience discomfort in breathing. The mechanism by which this blunted response to hypoxia occurs is unknown. We have previously shown that a computational model of the respiratory neural network (Diekman et al. in J Neurophysiol 118(4):2194-2215, 2017) can be used to test hypotheses focused on changes in chemosensory inputs to the central pattern generator (CPG). We hypothesize that altered chemosensory function at the level of the carotid bodies and/or the nucleus tractus solitarii are responsible for the blunted response to hypoxia. Here, we use our model to explore this hypothesis by altering the properties of the gain function representing oxygen sensing inputs to the CPG. We then vary other parameters in the model and show that oxygen carrying capacity is the most salient factor for producing silent hypoxemia. We call for clinicians to measure hematocrit as a clinical index of altered physiology in response to COVID-19 infection.

"The Psychiatric Domain of Post-Intensive Care Syndrome: A Review for the Intensivist".

Post-intensive care syndrome (PICS) is a clinical syndrome characterized by new or worsening changes in mental health, cognition, or physical function that persist following critical illness. The psychiatric domain of PICS encompasses new or worsened psychiatric burdens following critical illness, including post-traumatic stress disorder (PTSD), depression, and anxiety. Many of the established predisposing and precipitating factors for the psychiatric domain of PICS are commonly found in the setting of critical illness, including mechanical ventilation (MV), exposure to sedating medications, and physical restraint. Importantly, previous psychiatric history is a strong risk factor for the development of the psychiatric domain of PICS and should be considered when screening patients to diagnose psychiatric impairment and interventions. Delirium has been associated with psychiatric symptoms following ICU admission, therefore prevention warrants careful consideration. Dexmedetomidine has been shown to have the lowest risk for development of delirium when compared to other sedatives and has been the only sedative studied in relation to the psychiatric domain of PICS. Nocturnal dexmedetomidine and intensive care unit (ICU) diaries have been associated with decreased psychiatric burden after ICU discharge. Studies evaluating the impact of other intra-ICU practices on the development of the psychiatric domain of PICS, including the ABCDEF bundle, depth of sedation, and daily spontaneous awakening trials, have been limited and inconclusive. The psychiatric domain of PICS is difficult to treat and may be less responsive to multidisciplinary post-discharge programs and targeted interventions than the cognitive and physical domains of PICS. Given the high morbidity associated with the psychiatric domain of PICS, intensivists should familiarize themselves with the risk factors and intra-ICU interventions that can mitigate this important and under-recognized condition.

Factorial invariance of the Wechsler Intelligence Scale for Children, Fifth Edition, across the UK, US and Australia & New Zealand.

OBJECTIVES: This study examined the factorial invariance of the factor structure of the Wechsler Intelligence Scale for Children, Fifth Edition (WISC-V) across the UK, US and Australia & New Zealand (A&NZ). The factorial equivalence of cognitive assessments should be demonstrated before assuming cross-culture generalizability and interpretations of score comparisons. METHODS: Data were obtained from the UK, US and A&NZ normative standardizations of the WISC-V. The samples consisted of 415 UK, 2200 US and 528 A&NZ children, aged 6-16. Confirmatory factor analysis was applied separately in each sample to establish the baseline model. Next, tests of factorial invariance were undertaken using the recommended hierarchical approach, firstly across the UK and A&NZ samples and then across the UK and US samples. RESULTS: The five-factor first-order scoring model was found to be excellent fit across all three samples independently. Strict factorial invariance of the WISC-V was demonstrated firstly across the UK and A&NZ and secondly the UK and US nationally representative standardization samples. Comparison of latent means found small but significant differences in female children across the UK and A&NZ samples. CONCLUSIONS: Consistent with previous research, these results demonstrate the generality of the WISC-V factor structure across the UK, US and A&NZ. Furthermore, as the WISC-V factor structure aligns with the Cattell-Horn-Carroll (CHC) model of cognitive abilities, the results add further support to the cross-cultural generalizability of the CHC model. Small but significant differences in latent factor scores found across samples support the development and use of local normative data.

What is the Long-term Wear of a 5-Mrad Highly Crosslinked Polyethylene Liner? A 14-year RSA Study.

BACKGROUND: Lower doses of irradiation (≤ 5 Mrad) during the manufacture of highly crosslinked polyethylene acetabular liners may result in less crosslinking and an increased wear rate. Radiostereometric analysis (RSA) studies have found that wear of more highly irradiated highly crosslinked polyethylene liners (7 to 10 Mrad) decreases at longer-term follow-up compared with earlier reports of the same cohorts. Although wear of 5-Mrad irradiated liners appears to increase at midterm follow-up, it is unclear whether that remains true at longer follow-up. QUESTIONS/PURPOSES: We asked: (1) In patients who underwent THA with a 5-Mrad highly crosslinked polyethylene liner, what is the wear rate evaluated with RSA during the first 14 years? (2) Does the wear rate decrease after 6 years in situ? METHODS: This is a brief follow-up of prior RSA studies performed at 2 and 6 years. We prospectively reviewed the longer-term wear rate in 13 patients who underwent primary THAs with the same design of a 5-MRad irradiated crosslinked acetabular liner and a 28-mm cobalt-chromium articulation. Of the initial 30 patients who were enrolled, 13 (43%) were available at the 14-year timepoint; nine patients had died, 1 patient had withdrawn, 1 had an intraoperative fracture, 3 patients were too infirm to have radiographs, 2 had no baseline RSA radiographs, and 1 had poor-quality RSA images. Tantalum markers were inserted during surgery, and all patients had RSA radiographic examinations at 1 week, 6 months, and 1, 2, 6, and 14 years postoperatively. RESULTS: The mean ± standard deviation proximal, 2D, and 3D wear rates calculated between 1 year and 14 years were 0.019 ± 0.013 mm, 0.022 ± 0.015 mm, and 0.025 ± 0.019 mm per year, respectively. No patient had proximal 2D or 3D wear rates exceeding 0.06 mm per year. An increasing wear rate over time was measured for proximal and 2D wear rates between 6 and 14 years (0.024 and 0.030 mm per year) compared with that between 1 and 6 years (0.008 and 0.010 mm per year; p = 0.03). CONCLUSION: The proximal, 2D, and 3D wear of a highly crosslinked polyethylene liner produced using 5-Mrad radiation remains low in the longer-term. With the small numbers available in a long-term RSA study such as this, we confirmed that the wear rate did not decrease at longer-term follow-up, unlike previous RSA studies of more highly irradiated highly crosslinked polyethylene liners. Nevertheless, the wear rate remains very low and below the threshold typically associated with the development of osteolysis (0.1 mm/year of wear). This should provide assurance to orthopaedic surgeons monitoring patients with this 5-Mrad irradiated liner in situ, while providing useful information to manufacturers of future highly crosslinked polyethylene liners. LEVEL OF EVIDENCE: Level IV therapeutic study.

Increased Rates of Late Periprosthetic Fractures in Larger Hydroxyapatite-Coated Cementless Stems: Are Collared Stems a Better Alternative for Total Hip Arthroplasty?

BACKGROUND: Cementless hip stems are widely used for total hip arthroplasty (THA) and have demonstrated excellent survivorship. This study aimed to investigate the effects of stem size and calcar collars on rates of revision due to periprosthetic fracture. METHODS: All primary THA procedures recorded by the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR) from September 1999 to December 2021 for a diagnosis of osteoarthritis using a single cementless hip stem with modern bearings were included. The primary outcome measure was revision due to periprosthetic fracture. Stems were divided into 2 groups for comparison, large (size 14 to 20) and small-medium (6 to 13). A subanalysis was performed for collared stems. A total of 59,518 primary THA procedures were included. RESULTS: The cumulative percent revision for periprosthetic fracture was significantly higher for large stems compared to small-medium stems (hazard ratio [HR] = 1.57 [95% confidence interval {CI} 1.18, 2.09] P = .002). Furthermore, collared stems had significantly lower revision rates due to late periprosthetic fracture compared to collarless variants (2 week + HR = 4.55 [95% CI 3.23, 6.42], P < .001). Large collarless stems were found to have greater revision rates due to fracture compared to small-medium collarless stems (HR = 1.55 [95% CI 1.13, 2.12] P = .006), but no difference was found between collared groups (HR = 1.37 [95% CI 0.68, 2.78] P = .382). CONCLUSION: Large cementless hip stems have a higher rate of revision due to periprosthetic fracture compared to small-medium stems. Using a collared stem reduces the rate of periprosthetic fracture.

Carboxyl-terminal modulator protein (CTMP) deficiency mitigates denervation-induced skeletal muscle atrophy.

Denervated skeletal muscles show decreased Akt activity and phosphorylation, resulting in atrophy. Akt inhibits downstream transcription of atrophy-associated ubiquitin ligases like muscle ring-finger protein 1 (MuRF-1). In addition, reduced Akt signaling contributes to aberrant protein synthesis in muscles. In ALS mice, we recently found that carboxyl-terminator modulator protein (CTMP) expression is increased and correlated with reduced Akt signaling in atrophic skeletal muscle. CTMP has also been implicated in promoting muscle degeneration and catabolism in an in vitro muscle atrophy model. The present study examined whether sciatic nerve injury (SNI) stimulated CTMP expression in denervated skeletal muscle during muscle atrophy. We hypothesized that CTMP deficiency would reduce neurogenic atrophy and reverse Akt signaling downregulation. Compared to the unaffected contralateral muscle, wild-type (WT) gastrocnemius muscle had a significant increase in CTMP (p < 0.05). Furthermore, denervated CTMP knockout (CTMP-KO) gastrocnemius weighed more than WT muscle (p < 0.05). Denervated CTMP-KO gastrocnemius also showed higher Akt and downstream glycogen synthase kinase 3ß (GSK3ß) phosphorylation compared to WT muscle (p < 0.05) as well as ribosomal proteins S6 and 4E-BP1 phosphorylation (p < 0.001 and p < 0.05, respectively). Moreover, CTMP-KO mice showed significantly lower levels of E3 ubiquitin ligase MuRF-1 and myostatin than WT muscle (p < 0.05). Our findings suggest that CTMP is essential to muscle atrophy after denervation and it may act by reducing Akt signaling, protein synthesis, and increasing myocellular catabolism.

spatialGE: quantification and visualization of the tumor microenvironment heterogeneity using spatial transcriptomics.

SUMMARY: Spatially resolved transcriptomics promises to increase our understanding of the tumor microenvironment and improve cancer prognosis and therapies. Nonetheless, analytical methods to explore associations between the spatial heterogeneity of the tumor and clinical data are not available. Hence, we have developed spatialGE, a software that provides visualizations and quantification of the tumor microenvironment heterogeneity through gene expression surfaces, spatial heterogeneity statistics that can be compared against clinical information, spot-level cell deconvolution and spatially informed clustering, all using a new data object to store data and resulting analyses simultaneously. AVAILABILITY AND IMPLEMENTATION: The R package and tutorial/vignette are available at https://github.com/FridleyLab/spatialGE. A script to reproduce the analyses in this manuscript is available in Supplementary information. The Thrane study data included in spatialGE was made available from the public available from the website https://www.spatialresearch.org/resources-published-datasets/doi-10-1158-0008-5472-can-18-0747/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Efficient gradient boosting for prognostic biomarker discovery.

MOTIVATION: A gradient boosting decision tree (GBDT) is a powerful ensemble machine-learning method that has the potential to accelerate biomarker discovery from high-dimensional molecular data. Recent algorithmic advances, such as extreme gradient boosting (XGB) and light gradient boosting (LGB), have rendered the GBDT training more efficient, scalable and accurate. However, these modern techniques have not yet been widely adopted in discovering biomarkers for censored survival outcomes, which are key clinical outcomes or endpoints in cancer studies. RESULTS: In this paper, we present a new R package 'Xsurv' as an integrated solution that applies two modern GBDT training frameworks namely, XGB and LGB, for the modeling of right-censored survival outcomes. Based on our simulations, we benchmark the new approaches against traditional methods including the stepwise Cox regression model and the original gradient boosting function implemented in the package 'gbm'. We also demonstrate the application of Xsurv in analyzing a melanoma methylation dataset. Together, these results suggest that Xsurv is a useful and computationally viable tool for screening a large number of prognostic candidate biomarkers, which may facilitate future translational and clinical research. AVAILABILITY AND IMPLEMENTATION: 'Xsurv' is freely available as an R package at: https://github.com/topycyao/Xsurv. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optimal Generators for Quantum Sensing.

We propose a computationally efficient method to derive the unitary evolution that a quantum state is most sensitive to. This allows one to determine the optimal use of an entangled state for quantum sensing, even in complex systems where intuition from canonical squeezing examples breaks down. In this paper we show that the maximal obtainable sensitivity using a given quantum state is determined by the largest eigenvalue of the quantum Fisher information matrix (QFIM) and the corresponding evolution is uniquely determined by the coinciding eigenvector. Since we optimize the process of parameter encoding rather than focusing on state preparation protocols, our scheme is relevant for any quantum sensor. This procedure naturally optimizes multiparameter estimation by determining, through the eigenvectors of the QFIM, the maximal set of commuting observables with optimal sensitivity.

Volumetric measurement of intracranial meningiomas: a comparison between linear, planimetric, and machine learning with multiparametric voxel-based morphometry methods.

PURPOSE: To compare the accuracy of three volumetric methods in the radiological assessment of meningiomas: linear (ABC/2), planimetric, and multiparametric machine learning-based semiautomated voxel-based morphometry (VBM), and to investigate the relevance of tumor shape in volumetric error. METHODS: Retrospective imaging database analysis at the authors' institutions. We included patients with a confirmed diagnosis of meningioma and preoperative cranial magnetic resonance imaging eligible for volumetric analyses. After tumor segmentation, images underwent automated computation of shape properties such as sphericity, roundness, flatness, and elongation. RESULTS: Sixty-nine patients (85 tumors) were included. Tumor volumes were significantly different using linear (13.82 cm3 [range 0.13-163.74 cm3]), planimetric (11.66 cm3 [range 0.17-196.2 cm3]) and VBM methods (10.24 cm3 [range 0.17-190.32 cm3]) (p < 0.001). Median volume and percentage errors between the planimetric and linear methods and the VBM method were 1.08 cm3 and 11.61%, and 0.23 cm3 and 5.5%, respectively. Planimetry and linear methods overestimated the actual volume in 79% and 63% of the patients, respectively. Correlation studies showed excellent reliability and volumetric agreement between manual- and computer-based methods. Larger and flatter tumors had greater accuracy on planimetry, whereas less rounded tumors contributed negatively to the accuracy of the linear method. CONCLUSION: Semiautomated VBM volumetry for meningiomas is not influenced by tumor shape properties, whereas planimetry and linear methods tend to overestimate tumor volume. Furthermore, it is necessary to consider tumor roundness prior to linear measurement so as to choose the most appropriate method for each patient on an individual basis.

Tumor immune cell clustering and its association with survival in African American women with ovarian cancer.

New technologies, such as multiplex immunofluorescence microscopy (mIF), are being developed and used for the assessment and visualization of the tumor immune microenvironment (TIME). These assays produce not only an estimate of the abundance of immune cells in the TIME, but also their spatial locations. However, there are currently few approaches to analyze the spatial context of the TIME. Therefore, we have developed a framework for the spatial analysis of the TIME using Ripley's K, coupled with a permutation-based framework to estimate and measure the departure from complete spatial randomness (CSR) as a measure of the interactions between immune cells. This approach was then applied to epithelial ovarian cancer (EOC) using mIF collected on intra-tumoral regions of interest (ROIs) and tissue microarrays (TMAs) from 160 high-grade serous ovarian carcinoma patients in the African American Cancer Epidemiology Study (AACES) (94 subjects on TMAs resulting in 263 tissue cores; 93 subjects with 260 ROIs; 27 subjects with both TMA and ROI data). Cox proportional hazard models were constructed to determine the association of abundance and spatial clustering of tumor-infiltrating lymphocytes (CD3+), cytotoxic T-cells (CD8+CD3+), and regulatory T-cells (CD3+FoxP3+) with overall survival. Analysis was done on TMA and ROIs, treating the TMA data as validation of the findings from the ROIs. We found that EOC patients with high abundance and low spatial clustering of tumor-infiltrating lymphocytes and T-cell subsets in their tumors had the best overall survival. Additionally, patients with EOC tumors displaying high co-occurrence of cytotoxic T-cells and regulatory T-cells had the best overall survival. Grouping women with ovarian cancer based on both cell abundance and spatial contexture showed better discrimination for survival than grouping ovarian cancer cases only by cell abundance. These findings underscore the prognostic importance of evaluating not only immune cell abundance but also the spatial contexture of the immune cells in the TIME. In conclusion, the application of this spatial analysis framework to the study of the TIME could lead to the identification of immune content and spatial architecture that could aid in the determination of patients that are likely to respond to immunotherapies.