Clinical Research
Abstract
Metastatic prostate cancer is a clinically and molecularly heterogeneous disease. Under the selective pressure of androgen receptor (AR)–directed therapies, resistant phenotypes frequently emerge, posing significant diagnostic and therapeutic challenges. Neuroendocrine prostate cancer (NEPC) is a clinically important phenotype characterized by lineage plasticity, neuroendocrine features, visceral metastases and poor prognosis. Accurately diagnosing NEPC remains difficult due to its histologic and molecular complexity but has high clinical relevance. In this study, we developed a deep learning model that leverages interpretable cellular features to improve feature extraction from H&E-stained tissue sections (NEURAL-PC). By incorporating a multiple instance learning (MIL) framework, NEURAL-PC enables robust NEPC classification solely from H&E tumor images, achieving an area under the receiver operating characteristic curve (AUROC) of 0.921 in independent external validation. In addition to its diagnostic utility, NEURAL-PC provides prognostic information that enables further subclassification of advanced prostate cancer across diverse datasets supporting its strong prognostic value and generalizability. Broadly, our work highlights a hybrid approach that integrates features across different domains, offering a promising strategy for developing reliable deep learning tools in pathology. Built on this framework, NEURAL-PC represents an extensively validated diagnostic and prognostic model for advanced prostate cancer.
Authors
Zhijun Chen, Erolcan Sayar, Daniela Guevara, Helen Richards, Haoyue Zhang, Radhika A. Patel, Agnes C. Gawne, Lucas J. Liu, Ilsa Coleman, Ruth Dumpit, Colm Morrissey, Michael T. Schweizer, Ruben Raychaudhuri, Laura S. Graham, Evan Y. Yu, Heather H. Cheng, Chien-Kuang C. Ding, Yuzhuo Wang, Peter Choyke, Baris Turkbey, Chantal Chanel-Vos, Christina Fedorov, John R. Otilano III, Troy Kane, Jyothi Manohar, Michael Sigouros, Jones T. Nauseef, Ana Molina, David Nanus, Scott T. Tagawa, Juan Miguel Mosquera, Himisha P. Beltran, Ruth Etzioni, Peter S. Nelson, Rama Soundararajan, Ana M. Aparicio, Cora N. Sternberg, Michael C. Haffner, Stephanie A. Harmon
Abstract
Autoimmune Addison’s disease (AD) is a rare but life-threatening disorder caused by immune-mediated destruction of the adrenal cortex, and progress in therapy has been limited by insufficient mechanistic insight. Here, we establish a model of Experimental Autoimmune Adrenalitis (EAA) that recapitulates key features of AD and reveals sex-dependent differences in disease manifestation within the model. Immunization with peptides derived from the adrenal self-antigen CYP11A1 induces corticosterone insufficiency. We show that autoimmune adrenalitis is driven by IFNG produced by self-reactive CD4+ T cells, promoting granulomatous inflammation in the adrenal cortex. Together, these findings identify IFNG as a central effector of autoimmune adrenalitis and suggest that targeting the IFNG pathway may represent a potential therapeutic strategy for AD.
Authors
Arina Andreyeva, Juraj Michalik, Veronika Niederlova, Veronika Cimermanova, Ales Drobek, Radislav Sedlacek, Jan Prochazka, Juraj Labaj, Olha Fedosieieva, Waldemar Kanczkowski, Peter Draber, André Sulen, Ondrej Stepanek, Aleš Neuwirth
Abstract
BACKGROUND. Prior studies identified plasma proteins associated with chronic graft-versus-host disease (cGVHD). The goal of this cross-sectional study was to evaluate whether plasma biomarkers were associated with specific organ manifestations to help guide treatment choice. METHODS. Plasma proteins were measured in patients with cGVHD (n = 695) from Chronic GVHD Consortium studies. Correlations of plasma protein levels with individual organ involvement were tested with p≤0.05 considered significant after Benjamini-Hochberg adjustment and adjustment for five baseline clinical variables. RESULTS. Median time from cGVHD diagnosis to blood draw was 0.9 months (IQR 0.1–9.5). Donors were 50% HLA-matched unrelated, 32% matched related and the remainder were umbilical cord blood, haploidentical or mismatched unrelated donors. Methotrexate and calcineurin inhibitor acute GVHD (aGVHD) prophylaxis was used in 53% of patients. Overall, 326 (47%) had moderate and 244 (35%) had severe cGVHD with the following organ involvement at time of blood draw: skin (67%), mouth (60%), eye (49%), joint (34%), GI (31%), lung (23%), and liver (17%). After adjustment for batch effects and patient and transplant characteristics, fourteen plasma proteins were associated with organ involvement with independent AUCs of 0.7-0.8. All organs except eye were associated with at least one biomarker. However, no combination of plasma proteins improved model fit after adjusting for patient and transplant clinical variables. CONCLUSION. Correlations between plasma proteins and organ involvement were identified but are not actionable. Our future investigations will focus on more granular and immediately proximal determinants of cGVHD biology in both blood and tissue.
Authors
Stephanie J. Lee, Corey Cutler, Ningxin Ma, Timothy W. Randolph, George L. Chen, Joseph Pidala, Betty K. Hamilton, Carrie L. Kitko, Sally Arai, Najla El Jurdi, Lynn Onstad, Motoko Koyama, Catherine J. Lee, Sophie Paczesny, Geoffrey R. Hill
Abstract
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED/APS-1) is a monogenic autoimmune disorder of impaired central tolerance classically diagnosed by the presence of 2 out of 3 classic triad manifestations: chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency. However, many patients develop non-triad manifestations years earlier, delaying recognition and care. In 2016, we proposed expanded diagnostic criteria incorporating 3 early clinical manifestations — APECED rash, autoimmune enteritis, and enamel hypoplasia — based on observations in 35 North American patients. Here, we provide further support for the clinical utility of these expanded diagnostic criteria in independent cohorts of 57 American and 12 European patients enrolled in a prospective natural history study at the NIH. Across all cohorts, the expanded diagnostic criteria decreased the time to diagnosis by half relative to the classic diagnostic criteria. Patients exhibited an enrichment of early non-endocrine autoimmune manifestations, underscoring disease heterogeneity and the potential for developing organ-specific autoimmunity before endocrine failure. These findings demonstrate the clinical utility of the expanded APECED diagnostic criteria and support the notion that their adoption might enable earlier disease recognition and timely immunomodulatory therapy to improve long-term outcomes.
Authors
Elise M.N. Ferré, Joseph Pechacek, Monica M. Schmitt, Taura Webb, Heather Moorman, Thomas DiMaggio, Princess Barber, Vasielios Oikonomou, Stacey R. Rose, Peter D. Burbelo, Lindsey B. Rosen, Amy P. Hsu, Jennifer Stoddard, Shakuntala Rampertaap, Sergio D. Rosenzweig, Anjali Rai, Maria Teresa Magone, Chantal Cousineau-Krieger, Niki M. Moutsopoulos, Pamela J. Gardner, Heidi H. Kong, Leslie Castelo-Soccio, Ariane Soldatos, Katherine R. Calvo, Meryl Waldman, Behdad Afzali, Stefania Pittaluga, David Kleiner, Steven M. Holland, Kevin Fennelly, Jill Rothschild, Bryce A. Seifert, Magdalena Walkiewicz-Yvon, Theo Heller, Karen Winer, Michail S. Lionakis
Abstract
Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) has enhanced our understanding of host immune mechanisms in small cohorts, particularly in diseases with complex and heterogeneous immune responses such as sepsis. However, standard PBMC isolation from blood requires technical expertise and over two hours of onsite processing using Ficoll density gradient separation (‘Ficoll’) for scRNA-seq compatibility, precluding large-scale sample collection at most clinical sites. To minimize onsite processing, we developed Cryo-PRO (Cryopreservation with PBMC Recovery Offsite), a method of immediate onsite whole blood cryopreservation and subsequent batched PBMC isolation in a central laboratory prior to sequencing. We compared multimodal single-cell immune profiling results from samples processed using Cryo-PRO versus standard onsite Ficoll separation in 23 patients with sepsis. Critical outputs including cell substate fractions, marker genes, and surface protein expression were similar for each method across multiple cell types and substates, including an important monocyte substate enriched in patients with sepsis. Capture of T cell receptor transcripts was also comparable across both methods. Cryo-PRO reduced onsite sample processing time from >2 hours to <15 minutes and was reproducible across two enrollment sites, thus demonstrating potential for expanding multimodal single-cell analyses in multicenter studies of sepsis and other diseases.
Authors
Alyssa K. DuBois, Pierre O. Ankomah, Alexis C. Campbell, Renee Hua, Olivia K. Nelson, Christopher A. Zeuthen, M. Kartik Das, Shira Mann, Abigail Mauermann, Blair A. Parry, Nathan I. Shapiro, Michael R. Filbin, Roby P. Bhattacharyya
Abstract
Postnatal growth faltering is a pervasive problem among extremely preterm infants that is independently associated with adverse neurodevelopmental outcomes. We previously observed that preterm infants with poor postnatal growth have altered development of the intestinal microbiota relative to preterm infants with appropriate postnatal growth. Here, we used gnotobiotic mice to investigate whether these differences in microbiota development independently contribute to growth faltering. We found that colonization of neonatal mice with microbiotas from extremely preterm infants with poor growth reproduced postnatal growth impairment and induced a metabolic signature of enhanced lipolysis and fatty acid oxidation in the mice, characterized by elevated hepatic acylcarnitines and circulating ketones. In mice colonized at birth with microbiotas from infants with poor growth, postnatal treatment with microbiotas from infants with appropriate growth prevented growth impairment. These results indicate that altered development of the intestinal microbiota contributes to growth faltering in extremely preterm infants, and that microbiota modification can restore postnatal growth.
Authors
Kwai Tei Chan Poon, Se Hyang Han, Olga Ilkayeva, Michael J. Muehlbauer, Christopher B. Newgard, Charles M. Cotten, Patricia L. Ashley, Patrick C. Seed, John F. Rawls, Noelle E. Younge
Abstract
BACKGROUND. In vitro fertilization (IVF) culminates in embryo transfer into a hormonally primed endometrium, often via a programmed cycle (PC) regimen postulated to influence hypertensive disorders of pregnancy (HDP) risk. We thus generated a single-cell atlas of PC endometrium to define cell type-specific differences relative to natural cycle (NC) endometrium, and evaluated whether PC-associated modulation of the window of implantation (WOI) endometrium influences angiogenic balance in pregnancy. METHODS. Single-nucleus RNA-seq of prospectively collected PC and NC WOI endometrium. An independent prospective cohort of 548 singleton pregnancies was separately analyzed for maternal serum angiogenic markers (soluble fms-like tyrosine kinase-1; placental growth factor) and HDP incidence in PC- versus NC-conceived pregnancies, adjusting for clinical confounders and IVF use. RESULTS. Prominent transcriptomic differences were observed between PC (n = 7; 48,843 nuclei) and NC (n = 9; 44,230 nuclei) WOI endometrium, particularly in glandular epithelium (682 up- and 979 down-regulated genes; adjusted P < 0.05) and stromal fibroblasts (108 up- and 168 down-regulated). PC endometrium showed reduced uterine natural killer cell abundance, potentially from CXCL14 downregulation. Functional enrichment revealed downregulation of embryo implantation, angiogenesis, and extracellular matrix remodeling pathways in PC. Altered cell-cell signaling in decidualization, angiogenesis, and inflammatory response was also observed. Despite these WOI perturbations, PC-conceived pregnancies were not associated with early gestational angiogenic imbalance or increased HDP risk. CONCLUSION. PC endometrial preparation induced distinct cellular and signaling alterations in the WOI, but was not associated with subsequent development of angiogenic imbalance or HDP, thereby underscoring the resilience and adaptability of the early maternal-fetal interface. TRIAL REGISTRATION. ClinicalTrials.gov NCT03799107. FUNDING. ABOG/AAOGF; NICHD-R01-HD084380; NCTRI-P50-HD055764; NIAMS-P30-AR070155.
Authors
David Huang, Emily Flynn, Brittany R. Davidson, Juan C. Irwin, Mohammad Naser, Ana Laura Almonte, Jennifer Qin, Yue Song, Fleurdeliza B. Rabara, Rebecca Wong, Lydia B. Zablotska, Mitchell P. Rosen, Torsten Wittmann, Gabriela K. Fragiadakis, Alexis J. Combes, Marina Sirota, Marcelle I. Cedars, Linda C. Giudice
Abstract
Tuberous sclerosis complex (TSC) and Lymphangioleiomyomatosis (LAM) lack well-defined cellular origins, limiting treatment options. In this report, scRNA-seq of Tsc2+/− mouse renal cystadenomas revealed an 80-fold increase in a tumor cell subpopulation with neural crest features, and expressing known cranial neural crest genes as SRY box transcription factor 9 (Sox9), transcription factor activator protein (Tfap2a), and candidate neurocristopathy markers, osteopontin (Spp1), lipocalin-2 (Lcn2), clusterin (Clu), and cytokeratin 18 (Krt18). These signatures were validated in mouse tumors, and LAM patient lesions and serum, identifying a tumor phenotype distinct from traditional VEGFD detection. Pathway analysis indicated activation of WNT/SHH signaling, nephric duct formation, and pro-tumorigenic signals, with transcription factor 7 (Tcf7) and ephrin-A ligands as key upstream regulators. Spp1 KO in cranial neural crest cells (CNCCs) significantly reduced proliferation (28–33%), migration (54-76%), and invasion (29-64%) without affecting viability, while Tsc2 KO increased viability 3 to 6-fold with minimal impact on chemotaxis. Elevated serum levels of SPP1 and KRT18 in some LAM patients, decreased LCN2 in nearly all, and distinct increases in VEGFD suggest complementary roles for these biomarkers. Overall, findings support a neurocristopathic model of tumor development in TSC and LAM and identify potential biomarkers and therapeutic targets beyond mTOR inhibition.
Authors
Uchenna J. Unachukwu, Enio B. Garcia, Nooralam Rai, Jeanine M. D'Armiento
Abstract
Systemic lupus erythematosus (SLE) is a heterogeneous systemic autoimmune disease, yet the molecular basis underlying this variability remains incompletely understood. We profiled the plasma proteome in 260 SLE patients and 86 healthy volunteers (HVs) using the SomaScan v4.1 platform, quantifying 7,288 analytes corresponding to 6,595 unique proteins. We identified 215 proteins that were robustly differentially abundant between SLE patients and HVs in both discovery (n=207 SLE, n=45 HVs) and validation sets (n=53 SLE, n=41 HVs). Within-cases analyses identified 421 proteins associated with disease activity. Network-based clustering delineated correlated protein modules, including an interferon-associated module and a renal-associated module. Autoantibody-stratified analyses further uncovered distinct proteomic endotypes: positivity for antibodies targeting RNA-binding proteins (anti-Sm, anti-Ro-60, anti-RNP68, anti-RNP-A) was associated with increased interferon-stimulated protein levels (e.g., MX1, ISG15, CXCL10), independent of disease activity. Anti-Sm, anti-RNP-A and anti-Ro52 antibodies were associated with reduced plasma levels of their respective autoantigens. Anti-dsDNA antibodies were associated with elevated levels of CD40 ligand (CD40LG) and the neutrophil protease proteinase-3. Moreover, we identified an association between CD40LG and disease activity specific to the anti-dsDNA positive subgroup. Together, these data define plasma protein signatures of SLE and disease activity, highlight autoantibody-specific molecular phenotypes, and provide a basis for precision medicine.
Authors
Geoffrey H. D. Leung, Charlotte Bottomley, Norzawani Buang, Robert T. Maughan, Benjamin J. Whittle, Boroumand Zeidaabadi, Yun-Ju Huang, Tabitha Turner-Stokes, Marie Condon, Liz Lightstone, Tom Cairns, Marina Botto, Matthew C. Pickering, James E. Peters
Abstract
Cystic fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucus impairing the airway epithelia. Durable correction of these variants in airway cells remains a therapeutic challenge for about 10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant, 3120+1G>A, was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types resulting in notable enrichment of CFTR-expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited, but they decreased as a fraction of total cells and CFTR-expressing cells compared with unedited cells. CRISPR base editors delivered by polymeric nanoparticles (PNPs) facilitated functional rescue of CFTR to clinically meaningful levels in immortalized and primary airway cells. PNPs delivered GFP-encoding RNA to progenitor airway cells in fully differentiated airway cultures. Vitronectin was a major component of the PNP corona that formed in vivo, but preincubation with vitronectin did not enhance delivery. Together, these findings validate a scalable, nonviral platform with compelling translational promise for treating CF and other respiratory diseases involving respiratory epithelial cell dysfunction.
Authors
Erin W. Kavanagh, Anya T. Joynt, Audrey R. Pion, Alice C. Eastman, Alianna I. Parr, Katherine L. Starego, Manav Jain, Sydney R. Shannon, Edwin J. Yoo, Gregory A. Newby, Stephany Y. Tzeng, Neeraj Sharma, Jordan J. Green, Garry R. Cutting
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