Citations

Objective: To determine the prevalence of clinical significance reporting in contemporary comparative effectiveness research (CER). Background: In CER, a statistically significant difference between study groups may or may not be clinically significant. Misinterpreting statistically significant results could lead to inappropriate recommendations that increase health care costs and treatment toxicity. Methods: CER studies from 2022 issues of the Annals of Surgery , Journal of the American Medical Association , Journal of Clinical Oncology , Journal of Surgical Research , and Journal of the American College of Surgeons were systematically reviewed by 2 different investigators. The primary outcome of interest was whether the authors specified what they considered to be a clinically significant difference in the “Methods.” Results: Of 307 reviewed studies, 162 were clinical trials and 145 were observational studies. Authors specified what they considered to be a clinically significant difference in 26 studies (8.5%). Clinical significance was defined using clinically validated standards in 25 studies and subjectively in 1 study. Seven studies (2.3%) recommended a change in clinical decision-making, all with primary outcomes achieving statistical significance. Five (71.4%) of these studies did not have clinical significance defined in their methods. In randomized controlled trials with statistically significant results, sample size was inversely correlated with effect size ( r = −0.30, P = 0.038). Conclusions: In contemporary CER, most authors do not specify what they consider to be a clinically significant difference in study outcome. Most studies recommending a change in clinical decision-making did so based on statistical significance alone, and clinical significance was usually defined with clinically validated standards.

The diaphragm muscle is essential for breathing, and its dysfunctions can be fatal. Many disorders affect the diaphragm, including muscular dystrophies. Despite the clinical relevance of targeting the diaphragm, there have been few studies evaluating diaphragm function following a given experimental treatment, with most of these involving anti-inflammatory drugs or gene therapy. Cell-based therapeutic approaches have shown success promoting muscle regeneration in several mouse models of muscular dystrophy, but these have focused mainly on limb muscles. Here we show that transplantation of as few as 5000 satellite cells directly into the diaphragm results in consistent and robust myofiber engraftment in dystrophin- and fukutin-related protein-mutant dystrophic mice. Transplanted cells also seed the stem cell reservoir, as shown by the presence of donor-derived satellite cells. Force measurements showed enhanced diaphragm strength in engrafted muscles. These findings demonstrate the feasibility of cell transplantation to target the diseased diaphragm and improve its contractility.

ImportanceCancer is one of the leading causes of death in the United States, with the obesity epidemic contributing to its steady increase every year. Recent cohort studies find an association between bariatric surgery and reduced longitudinal cancer risk, but with heterogeneous findings.ObservationsThis review summarizes how obesity leads to an increased risk of developing cancer and synthesizes current evidence behind the potential for bariatric surgery to reduce longitudinal cancer risk. Overall, bariatric surgery appears to have the strongest and most consistent association with decreased incidence of developing breast, ovarian, and endometrial cancers. The association of bariatric surgery and the development of esophageal, gastric, liver, and pancreas cancer is heterogenous with studies showing either no association or decreased longitudinal incidences. Conversely, there have been preclinical and cohort studies implying an increased risk of developing colon and rectal cancer after bariatric surgery. A review and synthesis of the existing literature reveals epidemiologic shortcomings of cohort studies that potentially explain incongruencies observed between studies.Conclusions and RelevanceStudies examining the association of bariatric surgery and longitudinal cancer risk remain heterogeneous and could be explained by certain epidemiologic considerations. This review provides a framework to better define subgroups of patients at higher risk of developing cancer who would potentially benefit more from bariatric surgery, as well as subgroups where more caution should be exercised.

Background The rapid evolution of artificial intelligence (AI) in conjunction with recent updates in dual antiplatelet therapy (DAPT) management guidelines emphasizes the necessity for innovative models to predict ischemic or bleeding events after drug‐eluting stent implantation. Leveraging AI for dynamic prediction has the potential to revolutionize risk stratification and provide personalized decision support for DAPT management. Methods and Results We developed and validated a new AI‐based pipeline using retrospective data of drug‐eluting stent‐treated patients, sourced from the Cerner Health Facts data set (n=98 236) and Optum's de‐identified Clinformatics Data Mart Database (n=9978). The 36 months following drug‐eluting stent implantation were designated as our primary forecasting interval, further segmented into 6 sequential prediction windows. We evaluated 5 distinct AI algorithms for their precision in predicting ischemic and bleeding risks. Model discriminative accuracy was assessed using the area under the receiver operating characteristic curve, among other metrics. The weighted light gradient boosting machine stood out as the preeminent model, thus earning its place as our AI‐DAPT model. The AI‐DAPT demonstrated peak accuracy in the 30 to 36 months window, charting an area under the receiver operating characteristic curve of 90% [95% CI, 88%–92%] for ischemia and 84% [95% CI, 82%–87%] for bleeding predictions. Conclusions Our AI‐DAPT excels in formulating iterative, refined dynamic predictions by assimilating ongoing updates from patients' clinical profiles, holding value as a novel smart clinical tool to facilitate optimal DAPT duration management with high accuracy and adaptability.

Background Nephritis is a common manifestation of IgA vasculitis and is morphologically indistinguishable from IgA nephropathy. While MEST-C scores are predictive of kidney outcomes in IgA nephropathy, their value in IgA vasculitis nephritis has not been investigated in large multiethnic cohorts. Methods Biopsies from 262 children and 99 adults with IgA vasculitis nephritis ( N =361) from 23 centers in North America, Europe, and Asia were independently scored by three pathologists. MEST-C scores were assessed for correlation with eGFR/proteinuria at biopsy. Because most patients ( N =309, 86%) received immunosuppression, risk factors for outcomes were evaluated in this group using latent class mixed models to identify classes of eGFR trajectories over a median follow-up of 2.7 years (interquartile range, 1.2–5.1). Clinical and histologic parameters associated with each class were determined using logistic regression. Results M, E, T, and C scores were correlated with either eGFR or proteinuria at biopsy. Two classes were identified by latent class mixed model, one with initial improvement in eGFR followed by a late decline (class 1, N =91) and another with stable eGFR (class 2, N =218). Class 1 was associated with a higher risk of an established kidney outcome (time to ≥30% decline in eGFR or kidney failure; hazard ratio, 5.84; 95% confidence interval, 2.37 to 14.4). Among MEST-C scores, only E1 was associated with class 1 by multivariable analysis. Other factors associated with class 1 were age 18 years and younger, male sex, lower eGFR at biopsy, and extrarenal noncutaneous disease. Fibrous crescents without active changes were associated with class 2. Conclusions Kidney outcome in patients with biopsied IgA vasculitis nephritis treated with immunosuppression was determined by clinical risk factors and endocapillary hypercellularity (E1) and fibrous crescents, which are features that are not part of the International Study of Diseases of Children classification.

Despite notable advancements in treating chronic lymphocytic leukemia (CLL) with targeted therapies, a significant clinical challenge arises as a subset of CLL patients progress and develop Richter's transformation (RT), an aggressive form of lymphoma. While immune cell dysfunction and T cell defects are well-known characteristics of CLL, the alterations in immune cell subsets during CLL progression to RT remain largely unexplored. While CLL patients typically fail to respond to anti-PD1 pembrolizumab, a positive response has been observed in 20-40% of RT patients. Previously, our study involving three RT patients (1 responder and 2 progressors) revealed enriched regulatory T cells (Tregs) in the responders compared to progressors after pembrolizumab treatment (Iyer et al, ASH2020). Hence, we propose that dynamic changes in T cell subsets at baseline may be associated with response to pembrolizumab treatment. To this end, we performed single-cell RNA sequencing on sorted T cells (CD3 +) from PBMCs derived from 20 patients (responders (RES), n=5; stable disease (SD), n=5; progressive disease/progressors (PD), n=10). A total of 408,627 cells were captured, and an average of 1,393 genes were detected per cell per patient. Using a graph-based clustering approach (Seurat V4.3), we identified 19 clusters based on marker genes and transcriptional signatures and identified enriched pathways using Enrichr analysis. Our results showed that RES had an increased Th17 subset at baseline compared to PD (15% vs. 5%, p=0.01) and reduced Th2 T cells compared to SD (p=0.004) and PD (p=0.05). The Th17/Th2 ratio was higher in RES at baseline (Th17/Th2 R: 1.24, PD: 0.21, SD: 0.25, p0.05) and post-pembrolizumab (Th17/Th2 RES: 1.31, PD: 0.34, SD: 0.30, p0.05). Furthermore, RES had an increased fraction of proliferating CD4 T cells at baseline (4% RES vs. 0.4% SD and 0.4% PD, p=0.02) and post-treatment (1.9% RES vs. 0.8% PD and 0.34% SD, p=0.02). Although we observed an increasing trend in the fraction of Tregs in the RES at baseline [(7.6% RES vs.6.1% PD and 6.5% SD, p=0.4; ns) and post-treatment (10.3% RES vs. 8.3% PD and 5.8% SD, p= 0.2; ns] compared to PD and SD, the difference was not significant. Similarly, among CD8+ T cells, we observed an increasing trend in CD8+ effector T cell fraction in the responders at baseline (29.8% RES vs. 21% PD and 15.6% SD, p=0.9, ns) and post-treatment (30.1% RES vs. 21.2% PD and 19.9% SD, p=0.44, ns). However, the difference was not statistically significant. These findings highlight the dynamic changes in T cell subsets associated with pembrolizumab response. In terms of transcriptional changes in Tregs following pembrolizumab treatment among RES, PD, and SD, we observed dynamic changes on a few Treg marker genes, including TIGIT and TOX. TIGIT and TOX expression drastically decreased after treatment in RES (p < 0.0001), while expression did not change in PD and SD. There were no significant changes in the expression of FOXP3 or CD27 in RES, PD, and SD before and after the treatment. In CD8 effector T cells, GZMK, GZMA (granzymes), and PRF1 (perforin) are marker genes. We observed higher expression of GZMK and GZMA in RES (p <0.0001) at the baseline compared to SD and PD. In the proliferating CD4 T cells, we observed increased expression of IDH2 (Isocitrate dehydrogenase 2-key enzyme of TCA cycle) in RES at the baseline, as compared to PD and SD, indicating increased TCA cycle activity in the proliferating T cells. Pathway analysis of T cell clusters using the Enrichr database showed TNFA-IFNG-MYC targets are enriched in CD8 + cells, IL2-STAT5 signaling is upregulated in Th17, TREGs, and E2F cell cycle associated pathways enriched in CD4 + proliferation cluster. In conclusion, our study unveils significant baseline differences in CD4 subsets between RES and PD/SD subjects and dynamic changes in T cell subsets and transcriptional signatures. These findings offer potential predictors of pembrolizumab response in RT, such as increased Th17/Th2 ratio, enriched proliferating CD4 + T cells, and elevated TIGIT, TOX gene expression in Tregs, GZMK, and GZMA in CD8 effector cells. This deeper understanding of the immunological landscape in RT patients sheds light on the interplay between T cell subsets and treatment response in RT, paving the way for personalized therapeutic strategies. Further validation and exploration can lead to improved outcomes and tailored interventions for RT.

Introduction: Waldenström macroglobulinemia (WM) is a rare B-cell lymphoma that may be preceded by an asymptomatic IgM-monoclonal gammopathy of undetermined significance (MGUS). The mechanisms underlying the progression from IgM-MGUS to WM remain to be fully elucidated; however, the stepwise accumulation of genomic abnormalities has been suggested as a potential driver. Currently, the role of epigenetic regulation of DNA remains underexplored in this disease spectrum. Aberrant DNA methylation is a hallmark of oncogenesis that plays an important role in hematologic malignancies. The aim of this study was to assess differential DNA methylation in WM compared to IgM-MGUS and identify hyper- and hypomethylated pathways that may alter key cellular functions. Methods: This study included 15 patients with WM and 6 with IgM-MGUS. Bone marrow samples were prospectively collected and CD19+ and/or CD138+ B cells were positively selected. Subsequently, genome-wide DNA methylation analysis was performed. CpG methylation ratios were segmented into 200-bp regions and differentially methylated regions (DMRs) comparing WM to IgM-MGUS were identified. DMRs with a Q-value <0.05 and absolute methylation difference of ≥20% were considered significant and utilized for analysis. DMRs were annotated according to genomic regions of interest (promotors, 3'UTR, 5'UTR, introns, exons, CpG islands, CpG shores). Next, genes with significant promotor hyper- or hypomethylation were assessed using the Ingenuity Pathway Analysis. Pathways with a corrected p-value<0.05 were considered statistically significant and a Z-value was calculated based on the degree of differential methylation. Results: Genome-wide DNA methylation analysis revealed a significant number of DMRs (n= 15691, hyper: 8248; hypo: 7443) in WM compared to IgM-MGUS. Assessing genomic regions, promotors (hyper: 1379; hypo: 2403), introns (hyper: 1548; hypo: 2292), CpG shores (hyper: 587; hypo: 849) and 3'UTR (hyper: 66; hypo: 193) regions were observed to have a significantly greater number of hypomethylated DMRs (all comparisons, p<0.001) in WM compared to IgM-MGUS (Figure A). While exons (hyper: 1686; hypo: 1378), CpG islands (hyper: 2128; hypo: 82), and 5'UTR (hyper: 854; hypo: 246) regions had a significantly greater number of hypermethylated DMRs (all comparisons, p<0.001) (Figure A). Next, promotor methylation-based pathway analysis revealed differential methylation of multiple cellular pathways important in WM pathogenesis (Figure B). These included pathways involved in intracellular signaling and the regulation of cellular proliferation, such as the STAT3 and CXCR4 pathways which were hypomethylated, and the PTEN signaling pathway which was hypermethylated. Of interest, we have previously demonstrated the transcription factor, STAT3, to have an increased expression in WM and to be implicated in IgM secretion (Elsawa, 2011). Next, multiple cytokine signaling pathways were observed to be hypomethylated in WM compared to IgM-MGUS including members of the interleukin (IL) family (IL-1, IL-8, IL-15, IL-23), VEGF, and TGF-β. Of relevance, previous studies have demonstrated the elevation of IL-8, IL-15, and increased expression of angiogenic cytokines, including VEGF, in patients with WM (Lewicki, 2010; Anagnostopoulos, 2007). Furthermore, our group has also shown miRNA-based epigenetic regulation of IL-8 and IL-15 in WM compared to IgM-MGUS (Chohan, 2023). Lastly, several pathways critical in the regulation of the cell structure and the extracellular matrix (ECM) were found to be hypomethylated including the FGF, actin cytoskeleton, PAK, and FAK signaling pathways. Of note, the FGF/FGFR axis has been demonstrated to be dysregulated in WM, and its targeting has been shown to halt WM cell growth (Sacco, 2021). Conclusions: This study demonstrates differential DNA methylation of key genomic regions in WM compared to IgM-MGUS. Promotor methylation-based analysis reveals hypomethylation of several important cellular pathways involved in cytokine signaling, cell structure/ECM, and intracellular signaling. These results underscore the influence of epigenetics in the IgM-gammopathy disease spectrum and suggest the potential role of aberrant DNA methylation underlying the malignant progression from IgM-MGUS to WM.

Reprogramming of cellular metabolism is a hallmark of cancer leading to improved cellular fitness and providing a selective advantage to malignant cells. Recently, we found that follicular lymphoma (FL) patients with poor prognosis display increased transcriptional and protein expression of IRF4 (IRF4 high), altered immune signaling and an immunosuppressive tumor microenvironment (TME). Furthermore, high expression of IRF4 rewired B cell metabolism towards glycolysis ( Mondello P et al, Blood 2022;140 Suppl 1:168-169). However, it's unknown whether this metabolic reprogramming may be responsible in part for the impairment of immune cells in the TME, which also rely on glucose for their proliferation and function. Using RNA-seq on 88 FL patients, we found a negative correlation between IRF4-regulated genes associated with glycolysis and tumor infiltration by major immune cell subsets in IRF4 high compared to IRF4 low tumors ( Fig 1A), suggesting that IRF4-driven tumor glycolysis may reinforce an immune suppressed phenotype. Especially, we observed that ALDOA and GPI genes were inversely correlated with expression of CD4 + T cells (r=0.34, p=0.003; and r=0.38, p=0.015, respectively) and T reg cells (r=0.3, p=0.05; and r=0.35, p=0.027, respectively) in IRF4 high but not in IRF4 low tumors. As radioactive glucose is the cornerstone of PET assessment, we investigated the correlation between baseline SUV values and IRF4 expression. Remarkably, we found significantly higher levels of FDG uptake in FL IRF4 high compared to IRF4 low cases (p= 0.003), supporting the glycolytic features of Iymphoma expressing IRF4. To define the metabolic activity and function of lymphoma cells with different IRF4 status, we performed seahorse assay in TMD8 and SUDHL2 cells (lymphoma cells with IRF4 high expression) transfected with siIRF4 or siCtr for 72 hours. In line with the IRF4-dependence on glycolysis, TMD8 transfected with siCtr had a low baseline glycolytic ECAR, while the same value doubled after silencing IRF4. Notably, after adding oligomycin, which stimulates glycolysis, TMD8 control cells showed a lower glycolytic capability compared with those deprived of IRF4, probably due to the already maximal addiction to glycolysis ( Fig 1B left). We then investigated the glycolytic demand of germinal center (GC) B cells differently expressing IRF4. Using irf4 fl/fl mice ( Klein et al, Nat Immunol 2006) crossed with Cγ 1cre animals ( Cγ 1cre; irf4 fl/fl) to induce conditional deletion of irf4 in GC B cells, we tested ex-vivo sorted GC B cells from Cγ 1cre; irf4 fl/fl, Cγ 1cre; irf4 fl/+ and WT mice immunized for 10 days. This time point is critical for studying IRF4 as it corresponds to the GC reaction peak when GC B cells express elevated IRF4 levels promoting terminal differentiation to plasma cells. As expected, GC B cells minimally relied on glycolysis and showed a low ability to respond to oligomycin in WT mice ( Weiser et al, Nat Immunol 2020). However, GC B cells with partial or complete loss of irf4 demonstrated a superior ability to response to increased glycolytic demand ( Fig 1B right), further supporting the conclusion that IRF4 influences B cell dependency on glycolysis. As the malignant cell metabolism can alter nutrient availability in the TME, we explored if lymphoma cells differently expressing IRF4 may change glucose levels in the media and in turn impact T cells activity. We found low glucose and high lactate levels in the media of cell lines with IRF4 high expression, while the opposite was observed for those that were IRF4 low. We then cocultured primary human CD4 + T cells with TMD8 cells transfected with either siIRF4 or siCtr for 3 days at three different concentrations of D-glucose: high (22.2 mM, Gluc hi), normal (5.5 mM, Gluc nor) or absent (0 mM, Gluc nil). In line with our prior findings, coculture of B cells in which IRF4 was knocked out showed a significant decrease in abundance of T reg cells and an increase in T FH cells compared to control in presence of Gluc hi or Gluc nor. In contrast, there was no difference in the overall amount of either T cell subtype irrespective of IRF4 status with Gluc nil, supporting the prerequisite of microenvironmental glucose as primary fuel for T cell survival and function. In conclusion, IRF4 rewires the metabolism in B cells with profound functional implications on the TME. Our data suggest that glucose metabolism represents a functional vulnerability that might be exploited therapeutically.