Citations

Background: Flow cytometric detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is complex, time-consuming, and subject to inter-operator variability. Deep neural networks (DNNs) offer potential for standardization and efficiency improvement, but require rigorous validation and monitoring for safe clinical implementation. Methods: We evaluated a DNN-assisted human-in-the-loop approach for CLL MRD detection. Initial validation included method comparison against manual analysis (n = 240), precision studies, and analytical sensitivity verification. Post-implementation monitoring comprised four components: daily electronic quality control, input data drift detection, error analysis, and attribute acceptance sampling. Laboratory efficiency was assessed through a timing study of 161 cases analyzed by five technologists. Results: Method comparison demonstrated 97.5% concordance with manual analysis for qualitative classification (sensitivity 100%, specificity 95%) and excellent correlation for quantitative assessment (r = 0.99, Deming slope = 0.99). Precision studies confirmed high repeatability and within-laboratory precision across multiple operators. Analytical sensitivity was verified at 0.002% MRD. Post-implementation monitoring identified 2.97% of cases (26/874) with input data drift, primarily high-burden CLL and non-CLL neoplasms. Error analysis showed the DNN alone achieved 97% sensitivity compared to human-in-the-loop-reviewed results, with 13 missed cases (1.5%) showing atypical immunophenotypes. Attribute acceptance sampling confirmed 98.8% of reported negative cases were true negatives. The DNN-assisted workflow reduced average analysis time by 60.3% compared to manual analysis (4.2 ± 2.3 vs. 10.5 ± 5.8 min). Conclusions: The implementation of a DNN-assisted approach for CLL MRD detection in a clinical laboratory provides diagnostic performance equivalent to expert manual analysis while substantially reducing analysis time. Comprehensive performance monitoring ensures ongoing safety and effectiveness in routine clinical practice. This approach provides a model for responsible AI integration in clinical laboratories, balancing automation benefits with expert oversight.

Abstract RATIONALE: The alveolar epithelium is primarily comprised of two cell types – the flat alveolar type I (AT1) cell responsible for gas exchange, and the cuboidal AT2 cell responsible for surfactant secretion, alveolar defense and acts as facultative stem cells in injury repair. Both cell types emerge from a population of bipotent distal progenitors (DPs). While the FGF signaling involved in directing AT2 fate has been identified, the mechanism by which AT2 differentiation is precisely timed is not well understood. In embryonic stem cells, it has been demonstrated that differentiation is regulated by a biophysical parameter, cell membrane tension (CMT), that acts upstream of FGF signaling by suppressing receptor endocytosis. Given this, we sought to investigate whether and how CMT plays a role in timing the alveolar epithelial differentiation, particularly focusing on how CMT might regulate FGF signaling. METHODS: DPs were isolated from E15.5 mice and plated on top of or embedded in Matrigel. DPs were treated with small molecules, to rise CMT (CHIR22901 and Methyl Beta cyclodextrin; MBCD), inhibiting either endocytosis (Dynasore) or pathways downstream of FGF signaling (FR180204, Akt VIII, KRpep-2d, NSC 74859). CMT is measured both with optical trap technology and FlipTR dye, which uses fluorescence lifetime imaging microscopy. Tissues and cells were immunostained and imaged using markers for AT1 and AT2 cells, Fgfr2, and the endocytosis marker EEA1. The transgenic mice for this project are in a C57B6 strain background and include a pan-lung epithelial Cre (TgNkx2.1-Cre) bred to Cre-dependent ERK reporter (Rosa26SAE). RESULTS: We found that CMT reduces immediately prior to alveolar differentiation, and that increasing CMT with CHIR and MBCD blocks differentiation of DPs in culture. As CMT reduces, we observe an increase in endocytosis and internalization of Fgfr2 in both cultured cells and in vivo at E17.5. Further, scRNAseq analysis showed an upregulation of ERK target genes and we have validated higher ERK activity in nAT2s in the reporter line. Further, we validated that CMT regulates FGF signaling, as decrease in CMT results in more Fgfr2 endocytosis that likely activates ERK and Akt signaling. Our small molecule inhibition data suggests that ERK and Akt signaling are required for AT2 fate selection and we propose that they are the downstream targets of FGF signaling that are activated following lowered CMT and increased Fgfr2 endocytosis. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and fate selection during lung development.

Abstract RATIONALE: Alveoli form late in development and, for first breath, alveolar type 1 (AT1) as well as AT2 epithelial cell types must be functional. While prior studies have mapped the cellular and transcriptional programs underlying this process and identified AT fate inductive cues, questions remain as to the timing and regulation of their emergence. Thus, we sought to identify when, where, and how AT2 cells first arise and motivated by recent work in embryonic stem cells, we investigated whether the biophysical parameter cell membrane tension (CMT) plays a role. METHODS: Single-cell RNA-sequencing (scRNAseq) datasets sampling mouse lung development were processed using cluster correlation, velocity, and gene ontology analysis. Marker expression and AT2 morphology was assessed by immunostaining, and their migration confirmed by time-lapse confocal microscopy on embryonic precision-cut lung slices (PCLS). CMT was measured via optical tweezers (OT) on isolated cells as well as fluorescence lifetime (FLIM) on organoids and PCLS. Progenitors (E15.5) and AT2s (E16.5- >PN50) were isolated from distal region of the mouse lung, as well as human AT2s recently isolated, expanded, and banked in Mayo Clinic's recently established patient lung cell biorepository. CMT was raised using methyl-β-cyclodextran (MBCD) or polyethylene glycol (PEG). Small molecules CHIR and ICG-001 were used in culture to raise and lower β-catenin levels, respectively. Fluorescently labeled mesenchymal cells were isolated, expanded, and added to organoids in suspension for co-association experiments. RESULTS: No evidence of distal progenitors selecting either AT fate was found until E16.5, at which time we identified a distinct nascent AT2 (nAT2) state that ontology analysis detects upregulation of single-cell migration genes. Resistin-like molecule alpha (RELM-α), a type 2 inflammation regulator, labels nAT2s – which first individually emerged around E16.5 (although instances exist at E15.5) at intermediate zones between the branch tips and stalks of the distal epithelium. Following molecular emergence, we observed nAT2s extruded basally and can subsequently interact with nearby but anatomically distinct lumens, a behavior we provisionally name interlumenal junctioning and whose dynamics we validated by timelapse microscopy. Regarding CMT, we found its reduction in distal progenitors occurs prior to, and is required for, differentiation. We observed CMT reduction is sensitive to the manner of organoid culture, with on top culture resulting in more reduction and differentiation. Finally, we observed CMT is regulated cell-intrinsically via the adaptor protein β-catenin, as well as -extrinsically through osmotic (via PEG) or mesenchymal cell interactions, both of which reduce membrane fluidity.

Abstract Introduction Resilient smokers, who maintain respiratory health despite smoking, remain poorly characterized. Airway epithelial cell gene expression in resilient smokers can provide insight into mechanisms and pathways that may protect smokers from developing smoking-related lung disease. The objective of this study is to identify genes and potential pathways associated with lung-related resilience. Methods Resilient smokers were defined based on a previously published multi-dimensional definition which includes the absence of: 1) spirometric airflow limitation, 2) symptoms (cough/sputum, dyspnea), 3) radiographic (HRCT) abnormalities, 4) exacerbations, and 5) an accelerated FEV1 decline. “Non-resilient” smokers had preserved spirometry but showed abnormalities in at least one domain of resilience. We analyzed airway epithelial brush samples from 21 resilient and 34 non-resilient smokers from SPIROMICS. Differential gene expression was performed using DESeq2 package in R, which estimates differential expression using generalized linear models. We adjusted for sequencing batch, age (continuous), race, gender, and current smoking status. While controlling for these covariates we evaluated differential gene expression between resilient and non-resilient smokers, the interaction between resilience and smoking status, and in each sub-category of resilient (absence of symptoms, radiographic abnormalities, exacerbations, and accelerated decline in FEV1). Results We identified 98 genes that were differentially expressed (FDR ≤0.1) when comparing resilient to non-resilient smokers, 20 of those genes were up regulated in resilient smokers (Figure 1). Notable genes included cadherin-12 (CDH12), aspartate beta-hydroxylase (ASPH), and protein kinase AMP-activated alpha 2 (PRKAA2). CDH12 is involved in cell adhesion and epithelial integrity. ASPH influences cell adhesion and signaling. PRKAA2 plays role in cellular homeostasis and stress response, which may be altered in non-resilient smokers, affecting cellular metabolism and inflammation. BPI folding containing family A member (BPIFA1) was significantly associated with smoking status in the interaction analysis, suggesting its role in modulating the impact of smoking on resilience. BPIFA1 has anti-microbial properties, regulates immune responses, and contributes mucosal surface protection. Sub-category analysis identified 17 genes associated with resilience to symptoms and 2 genes related to physiologic resilience (FEV1 decline), though no significant genes were associated with radiographic or exacerbation resilience. Conclusion Differential gene expression of airway epithelial cells identified distinct expression patterns between resilient and non-resilient smokers. Specifically, genes associated with epithelial cell adhesion, signaling, and cellular homeostasis were up regulated in resilient smokers. These findings suggest that resilient smokers may have transcriptomic profiles and potential mechanisms that mitigate the effects of cigarette smoke.

Abstract Rationale: MUC5B and MUC5AC are neighboring genes on chromosome 11p15 that encode the two predominant mucins secreted in the respiratory airways. Increased airway mucin concentrations and an imbalance between these two mucins drive COPD symptoms and the development of airflow obstruction or COPD, but the epigenomic regulation of this loci has not been characterized in the airways. Methods: We isolated DNA from bronchial airway brushings of 72 participants with >20 pack-years smoking history from the Subpopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS). 36 participants had GOLD-based moderate-to-severe COPD (FEV1/FVC<0.70, FEV1<80%) and 36 were without or with mild COPD. We used the Illumina Infinium Methylation EPIC BeadChip Array to assess proportion of DNA methylation (β) spanning 689,937 CpG sites. We evaluated proportional DNA methylation (β) at 96 CpG loci within a 103kB region using matrixQTL for cis-expression quantitative trait methylation (eQTM: 10kB flanks relative to MUC5AC/MUC5B) integrating bulk RNA-seq data and mQTL models integrating gene sequencing data to identify DNA variation associated with proportion eQTM methylation. Models included age, sex, BMI, clinical site, current smoking vs past smoking, total pack-years, three ancestral components of ancestry, chipset, and position. Results: We identified a top eQTM, cg00524486, marginally associated with MUC5B expression (coefficient estimate=8.75, punadjusted=0.0014, pFDR=0.065, Figure 1B). This eQTM was associated with CT scan parametric response mapping-based functional small airways disease (PRM-fSAD, beta coefficient[β]=3.64, p=0.011). We tested for DNA variation associated with methylation of this top eQTM which identified two methylation quantitative trait loci (mQTL: rs885454 [β=0.044, minor allele frequency[MAF]=0.09, pFDR=0.01], rs868902 [β=0.020, MAF=0.43, pFDR=0.08, Figure 1A) associated with cg00524486 methylation, of which the more frequent mQTL, rs868902, was marginally associated with PRM-fSAD (p=0.07) in the larger SPIROMICS cohort with MUC5B DNA sequencing data. Conclusions: This preliminary multi-omic study of the lower airway epithelium demonstrates that the expression of MUC5B is epigenetically regulated by neighboring CpG probe methylation (eQTM) represented by DNA variation (mQTL). These studies require further confirmation in a larger dataset of airway samples and support a role for epigenetic variation as a biologic contributor and biomarker of early COPD pathogenesis.

Abstract RATIONALE: Airway-to-lung ratio (AirLR), calculated as the mean of airway lumen diameters at standard anatomic locations divided by the cube-root of total lung volume, is associated with COPD risk regardless of smoking status, and we have previously shown an association between total-pulmonary-vascular-volume-to-lung-size relationship with AirLR. [PMCID: PMC11284327] Using dual energy computed tomography (DECT), we recently developed a deep learning-based algorithm for pulmonary arterial segmentation to compute pulmonary-arterial-to-lung ratio (ArtLR) which correlated significantly with AirLR and markers COPD. Here we automate image processing in a larger cohort to estimate ArtLR, introduce an alternative ArtLR standardization and correlate it to COPD-related measures of lung structure and function. METHODS: Using a SPIROMICS sub-cohort (110 participants), imaged via contrast enhanced DECT at functional residual capacity (FRC), an automatic deep learning pipeline segmented the arteries from the pulmonary vascular tree. Another pipeline automatically extracted the centerlines and calculated average arterial segment diameters. A paired non-contrast total lung capacity (TLC) scan was registered to the DECT FRC scan to transfer airway segment labels from the TLC to FRC domain. AirLR was calculated as the mean of airway lumen diameters at standard anatomic locations (trachea-to-subsegments) divided by cube root of lung volume [TLC or FRC]. A simple Long Short-Term Memory (LSTM) network was used to find corresponding anatomic arterial segments. Associations between ArtLR and %Emphysema, AirLR, pre- and post-bronchodilator FEV1/FVC, and a texture-based (AMFM) measure of %broncho-vascular bundles was assessed using linear regression to adjust for age, sex and BMI. RESULTS: In adjusted analyses, a 1-SD decrement in ArtLR-TLC was associated with 4.198 units increase in %Emphysema-910 (95%CI: 1.360-7.037; p = 0.0041), and 0.8198 units decrease in %Bronchovascular (95%CI: 0.5037-1.136; p < 0.0001). A 1-SD increment in ArtLR-TLC was associated with 0.2515 SD increase in AirLR-Outer (95%CI: 0.04947SD-0.4536SD; p = 0.0152), and 0.02083 units increase in PreBronch-FEV1FVC (95%CI: 0.001814-0.03984; p = 0.0321). Sex and BMI show significant associations with ArtLR-TLC, where female is associated with 1.068 SD decrease in ArtLR, and a unit increase in BMI is associated with 0.05788 SD increase in ArtLR (both p-values < 0.0001), and age was not association with ArtLR. CONCLUSIONS: An automated pipeline for pulmonary arterial tree segmentation and standardization from contrast enhanced DECT FRC scans demonstrates a significant relationship between ArtLR and AirLR as well as between ArtLR and %emphysema, and FEV1/FVC. A standardized method of arterial tree size assessment provides for expanded exploration of vascular vs airway disease etiologies.

Abstract Rationale Chronic obstructive pulmonary disease (COPD) manifests later in life and is a leading cause of death worldwide. While smoking-related lung pathology is a long-recognized COPD etiotype, emerging evidence suggests that early-life growth conditions also play an important role. A recently-developed index of early-life growth conditions – the difference between attained height and genotype-predicted height (height-GaP) – has been validated against prospectevely ascertained adverse early-life growth conditions. This study used height-GaP to evaluate the association of early-life growth conditions with COPD phenotype and prognosis later in life. Methods Among current and former smokers with spirometry-defined COPD in the SubPopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS), we computed height-GaP as the difference between attained height and genotype-predicted height. Regression models were fit for respiratory mortality (defined by primary diagnostic code), dyspnea prevalence (Medical Research Council dyspnea rating >1), chronic bronchitis prevalence, airflow obstruction severity as quantified by FEV1Q, and functional capacity (six-minute walk distance), adjusting for age, age2, sex, race-ethnicity, genotype-predicted height, cigarette smoking status and pack-years. Sensitivity analyses accounted for competing risks of death (Fine-Gray subdistribution hazard model) and accounted for later-life height loss (restricting sample to participants under 50 years). Results Among 1,258 participants with COPD (mean±SD age: 65.4±8.0 years, 42.4% female, mean±SD pack-years: 53.6±28.2, mean±SD FEV1%: 63.3±23.9%), there were 202 respiratory deaths over a median 7.5 years of follow-up (Figure). Larger height-GaP deficit was associated with higher respiratory mortality (adjusted hazard ratio [aHR]: 1.30 [95%CI: 1.12 to 1.50] per 1-SD increment in height-GaP deficit). This association remained significant after accounting for competing events (p=0.001) and when restricted to participants under 50 years (p=0.032). Larger height-GaP deficit was associated with higher dyspnea prevalence (adjusted prevalence ratio: 1.13 [95%CI: 1.02 to 1.25] per 1-SD increment in height-GaP deficit), lower FEV1Q (adjusted mean difference: -0.37 [95%CI: -0.45 to -0.29] per 1-SD height-GaP deficit), and shorter 6-minute walk (adjusted mean difference: -16.6 m [95%CI: -23.1 m to -10.0 m] per 1-SD increment in height-GaP deficit), but not chronic bronchitis or CAT score ≥10 (p>0.05 for each). Conclusions Among current and former smokers with COPD, a quantitative index of early-life conditions was associated with higher respiratory mortality, in addition to dyspnea, airflow obstruction severity and reduced functional capacity. These results suggest that early-life growth conditions may influence COPD phenotype and prognosis later in life.

Abstract RATIONALE: The alveolar epithelium consists of two cell types (AT2 and AT1) derived from a common Distal Progenitor (DP). Understanding how the epithelium of alveoli functions and becomes differentiated is essential for treatment of lung diseases. One possible regulator of cell fate is cell membrane tension (CMT), a biophysical property measured by the energy required to deform the membrane and known to be an important regulator of cell movement and endocytic processes. AT1 fate selection is largely driven and reinforced by YAP (Yes-associated protein), a mechano-sensitive transcription factor gated from entering the nucleus by the size of nuclear pores, which increases upon stretch. Here, we investigated whether and how CMT regulates the transduction of stretch-dependent YAP signaling and the remodeling into AT1 cells. METHODS: DPs were isolated from E15.5 mice and cultured on Matrigel for use in differentiation experiments. Immunostaining for AT1 and AT2 markers, Phalloidin, DAPI and YAP were used to assess YAP localization and architectural changes. We generated transgenic mice containing a pan-epithelial Cre (TgNkx2.1-Cre), a Cre-dependent driver of ltta (TigreAi140), and a TetOff regulated construct expressing YAP1 (Rosa26teto-Yap1), resulting in constitutive Yap1 expression in Cre-expressing DPs. CMT is measured both with optical trap technology and with the use of FlipTR dye, which uses Fluorescence Lifetime Imaging Microscopy (FLIM) to determine CMT. RESULTS: To demonstrate the role of CMT in timing AT1 fate, we found that CMT lowers in DPs concomitantly with differentiation, and that flattening cells (likely AT1s) have even lower CMT than round cells (likely AT2s). Using organotypic culture, we found that raising CMT with MBCD, PEG, or Matrigel embedding largely stops differentiation of DPs. Surprisingly, even when YAP is overdriven in mice in E15.5 distal progenitors, early precocious AT1 differentiation is not observed, suggesting YAP signaling is not alone sufficient to drive AT1 fate selection in DPs with higher CMT. We reproduced this finding when culturing DPs under high CMT conditions-promoting high YAP/TAZ levels via LATS inhibition was also not sufficient to select for AT1 fate. Finally, we found that high CMT disrupts nuclear YAP localization, nuclear-actin attachment, and cytoskeletal remodeling that is typically when DPs remodel towards AT1 fate. We propose that high baseline CMT in DPs prevents cellular remodeling, specifically that it suppresses the formation of actin-nuclear associations that imbue the nucleus with stretch sensitivity, thereby preventing an increased sensitivity to stretch and thus higher YAP signaling.

Objective To develop and validate classification criteria for pediatric chronic nonbacterial osteomyelitis (CNO) jointly supported by EULAR and the American College of Rheumatology (ACR). Methods This international initiative had 4 phases: (1) candidate items were proposed in a survey of pediatric rheumatologists, (2) criteria definition and reduction by Delphi and nominal group technique exercises, (3) criteria weighting using multicriteria decision analysis, and (4) refinement of weights and threshold score in a development cohort of 441 patients and validation in another cohort of 514 patients. Results The new EULAR/ACR classification criteria for CNO require typical radiographic or magnetic resonance imaging findings and bone pain as an obligatory entry criterion and exclusion criteria of malignancy, infection, vitamin C deficiency, and hypophosphatasia, followed by additive weighted criteria in 5 clinical (site of bone lesions, pattern of bone lesions, age at onset, coexisting conditions, fever) and 4 pathology/laboratory domains (bone biopsy findings if done, anemia, C‐reactive protein level, and erythrocyte sedimentation rate). A total score ≥55 is required for classification as CNO. The new criteria had a sensitivity of 82% and specificity of 98% in the validation cohort. Conclusion These new classification criteria for pediatric CNO developed with international input reflect current views about CNO, have high specificity and good sensitivity, and provide a key foundation for future CNO research.

Background We hypothesized treatment with nivolumab and stereotactic radiosurgery (SRS) would be feasible, well tolerated, and may improve intracranial tumor control over SRS alone for breast cancer brain metastases (BCBM). Methods The study is a phase Ib trial of nivolumab and SRS for BCBM. Clinical trial information: NCT03807765 . Key eligibility criteria include BCBM of all subtypes, age ≥18, Eastern Cooperative Oncology Group Performace Status (ECOG-PS)≤2 with ≤10 brain metastases. Treatment was initiated with a dose of nivolumab (480 mg intravenously) that was repeated every 4 weeks. The initial dose of nivolumab was followed 1 week later by SRS. Blood was collected at baseline and every 4 weeks for flow cytometry and cell-free DNA (cfDNA) assessment. Results A total of 12 patients received SRS to 17 brain metastases. Breast cancer subtypes included triple negative (50%), hormone receptor (HR)+/HER2− (33%), and HR−/HER2+ (17%). Median follow-up from start of protocol therapy is 56 months. No cases of radionecrosis were noted. Two lesions were noted to undergo local failure, both pathologically confirmed, for a 12-month local control of 94%. Median distant intracranial control was 7.4 months with a 12-month control rate of 33%. Median systemic progression-free survival was 7.7 months with a 12-month rate of 42%. Median overall survival (OS) was 24.7 months with a 12-month OS of 75%. Most patients were noted to have an increase in cfDNA throughout study treatment, at week 5 compared with baseline (83%), week 25 compared with baseline (89%), and 100% at first follow-up. Intracranial control was associated with lower levels of CD4 regulatory T cells (Treg) (p=0.03) and higher levels of CD4 T effector memory (p=0.04). Conclusions Nivolumab and SRS is a safe and feasible treatment option in BCBM. Long-term follow-up revealed no cases of radiation necrosis. Trial registration number NCT03807765 .