Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus.

BACKGROUND: Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA-sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality. OBJECTIVES: We sought to comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (ie, eosinophilic esophagitis [EoE]). METHODS: We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings. RESULTS: In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n = 6 individuals] and 398 from esophagus [n = 6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably IFN-γ, IL-10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and the hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DNAX-activation protein-12 (also known as TYROBP) interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling. CONCLUSIONS: In EoE, esophageal eosinophils exist as 2 populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types.

Comprehensive genomic profiling of ESR1, PIK3CA, AKT1, and PTEN in HR(+)HER2(-) metastatic breast cancer: prevalence along treatment course and predictive value for endocrine therapy resistance in real-world practice.

BACKGROUND: The treatment landscape for HR(+)HER2(-) metastatic breast cancer (MBC) is evolving for patients with ESR1 mutations (mut) and PI3K/AKT pathway genomic alterations (GA). We sought to inform clinical utility for comprehensive genomic profiling (CGP) using tissue (TBx) and liquid biopsies (LBx) in HR(+)HER2(-) MBC. METHODS: Records from a de-identified breast cancer clinicogenomic database for patients who underwent TBx/LBx testing at Foundation Medicine during routine clinical care at ~ 280 US cancer clinics between 01/2011 and 09/2023 were assessed. GA prevalence [ESR1mut, PIK3CAmut, AKT1mut, PTENmut, and PTEN homozygous copy loss (PTENloss)] were calculated in TBx and LBx [stratified by ctDNA tumor fraction (TF)] during the first three lines of therapy. Real-world progression-free survival (rwPFS) and overall survival (rwOS) were compared between groups by Cox models adjusted for prognostic factors. RESULTS: ~ 60% of cases harbored 1 + GA in 1st-line TBx (1266/2154) or LBx TF ≥ 1% (80/126) and 26.5% (43/162) in LBx TF < 1%. ESR1mut was found in 8.1% TBx, 17.5% LBx TF ≥ 1%, and 4.9% LBx TF < 1% in 1st line, increasing to 59% in 3rd line (LBx TF ≥ 1%). PTENloss was detected at higher rates in TBx (4.3%) than LBx (1% in TF ≥ 1%). Patients receiving 1st-line aromatase inhibitor + CDK4/6 inhibitor (n = 573) with ESR1mut had less favorable rwPFS and rwOS versus ESR1 wild-type; no differences were observed for fulvestrant + CDK4/6 inhibitor (n = 348). CONCLUSION: Our study suggests obtaining TBx for CGP at time of de novo/recurrent diagnosis, followed by LBx for detecting acquired GA in 2nd + lines. Reflex TBx should be considered when ctDNA TF < 1%.

Model of perinatal care but not prenatal stress exposure is associated with birthweight and gestational age at Birth: The Australian birth in the time of COVID (BITTOC) study.

OBJECTIVE: The present study aimed to understand, relative to standard care, whether continuity of care models (private midwifery, continuity of care with a private doctor, continuity of care with a public midwife), and women's experience of maternity care provision, during the perinatal period buffered the association between prenatal maternal stress (PNMS) and infant birth outcomes (gestational age [GA], birth weight [BW] and birth weight for gestational age [BW for GA]). METHODS: 2207 women who were pregnant in Australia while COVID-19 restrictions were in place reported on their COVID-19 related objective hardship and subjective distress during pregnancy and provided information on their model of maternity care. Infant birth outcomes (BW, GA) were reported on at 2-months postpartum. RESULTS: Multiple linear regressions showed no relationship between PNMS and infant BW, GA or BW for GA, and neither experienced continuity of care, nor model of maternity care moderated this relationship. However, compared with all other models of care, women enrolled in private midwifery care reported the highest levels of experienced continuity of care and birthed infants at higher GA. BW and BW for GA were higher in private midwifery care, relative to standard care. CONCLUSION: Enrollment in continuous models of perinatal care may be a better predictor of infant birth outcomes than degree of PNMS exposure. These results highlight the possibility that increased, continuous support to women during pregnancy may play an important role in ensuring positive infant birth outcomes during future pandemics.

Adaptations to sociality in the mimetic and auricular musculature of the African wild dog (Lycaon pictus).

The African wild dog (Lycaon pictus) is a highly social canid that engages in sophisticated, coordinated group hunting tactics to procure large game. It is one of the most effective hunters of the African savannah, due to its highly developed communication methods. It also has large, mobile ears which enhance its auditory capabilities while hunting and assist with thermoregulation. Recent research suggested that certain muscles of facial expression, particularly those involved with expressive eyebrow movement, evolved solely in domestic dogs (Canis familiaris) to facilitate communication with their human owners. However, it is unclear whether highly social wild canid species may also employ similar expressive eye communication. We performed detailed dissections of an adult male L. pictus to evaluate and describe its mimetic and auricular musculature. Overall, L. pictus has well-developed facial and ear muscles. Musculi levator anguli oculi medialis (LAOM) and retractor anguli oculi lateralis (RAOL), mimetic muscles of hypothesized importance in domestic dog-human non-verbal communication, are enlarged in L. pictus, comparable in size to those of domestic dogs, as is m. orbicularis oculi. This morphology suggests that ocular facial expressions contribute to within-pack communication in wild dogs and are not unique to domestic dogs. The auricular muscles of L. pictus are well-developed, supporting greater leverage and fine manipulation of its large, mobile ears. These muscular adaptations facilitate the highly social ecology of African wild dogs and challenge current interpretations about the unique nature of domestic dog facial expressions.

Melanoma-like features in pediatric longitudinal melanonychia: A systematic review and meta-analysis.

BACKGROUND: Pediatric longitudinal melanonychia (LM) can exhibit atypical features that mimic red-flag signs for subungual melanoma in adults and lead to diagnostic uncertainty. Nail biopsy may be unnecessary if clinical inspection and dermoscopy suggest a benign nature. METHODS: We searched PubMed and Embase from inception to February 2023 for studies of any design reporting either the number or proportion of clinical and dermoscopic features in at least five children (≤18 years) with LM. Non-English articles, reviews, and abstracts were excluded. We performed a systematic review and meta-analysis to collate all existing data. RESULTS: A total of 1218 articles were screened and 24 studies with 1391 pediatric patients were included. Nevus was the most common diagnosis (86.3%). The most prevalent sites were fingernails (76.2%) and first digits (45.4%). Pooled proportions of common features were: dark-color bands (69.8%), multi-colored bands (47.6%), broad bandwidth (41.1%), pseudo-Hutchinson sign (41.0%), irregular patterns (38.1%), Hutchinson sign (23.7%), dots and globules (22.5%), nail dystrophy (18.2%), and triangular sign (10.9%). Outcomes included progression (widening or darkening, 29.9%), stability (23.3%), and spontaneous regression (narrowing or fading, 19.9%). Only eight cases of subungual melanoma in situ were reported, and no invasive melanomas were identified. CONCLUSION: Although atypical characteristics are common in pediatric LM, the probability of malignant transformation is exceedingly low. Appropriate evaluation and management of pediatric LM includes careful clinical and dermoscopic inspection with attention to benign features followed by long-term interval follow-up.

Gestational vitamin D and offspring fracture risk: do associations persist into mid adolescence?

BACKGROUND: Previous studies report that maternal vitamin D exposure during pregnancy is associated with offspring later-life bone health. A study in the Vitamin D in Pregnancy (VIP) cohort reported sexually dimorphic effects of maternal 25-hydroxyvitamin-D (25(OH)D) and offspring fracture profiles at 10 years of age. We, therefore, aimed to determine associations between maternal 25(OH)D status and offspring fracture risk at 16 years of age in this cohort. METHODS: In total, 475 mother-child pairs were recruited to the VIP study in southeastern Australia. Maternal serum samples were obtained at recruitment (<16 weeks' gestation) and/or 28-32 weeks' gestation and analysed for 25(OH)D. Radiologically-confirmed incident fractures in children were ascertained from date of birth (2002-2004) until July 16, 2019. Cox proportional hazard models were used to determine associations between maternal 25(OH)D and childhood fracture risk, and final models included maternal age at recruitment, offspring sex, birth weight, gestation length and season of 25(OH)D sample. RESULTS: Data were available for 400 children (mean age 16.1 years). There were 122 (30.5%) children who sustained at least one fracture. Higher maternal 25(OH)D (per 10 nmol/L) in early gestation was associated with a decreased fracture risk in boys (HR 0.87; 95% CI: 0.77, 0.99); the pattern was reversed in girls (HR 1.10; 95% CI 1.00, 1.22). At late gestation, higher maternal 25(OH)D was associated with an increased fracture risk in girls (HR 1.14; 95% CI: 1.04, 1.24). CONCLUSIONS: While our findings must be interpreted within the constraints of our limitations, we report that the contradictory risk profiles observed at early childhood in this cohort remain in adolescence.

Relationships between cortisol levels across early childhood and processing speed at age 4.5 years in children born very preterm.

Children born very low gestational age (VLGA, 29-32 weeks gestational age [GA]) display slower processing speed and altered hypothalamic pituitary adrenal (HPA) axis function, with greater effects in those born extremely low gestational age (ELGA; 24-28 weeks GA). We investigated trajectories of HPA axis activity as indexed by cortisol output and patterns across cognitive assessment at ages 1.5, 3 and 4.5 years, comparing children born ELGA and VLGA and associations with 4.5-year processing speed. In a prospective longitudinal cohort study, infants born very preterm (<33 weeks gestation) returned for developmental assessment at ages 1.5, 3, and 4.5 years. At each age, children completed standardized cognitive testing and saliva samples collected before (Pretest), during (During) and after (End) challenging cognitive tasks were assayed for cortisol. For the total group (n = 188), cortisol area under the curve with respect to ground (AUCg) decreased, while cortisol reactivity to challenge (Pre-test to During) increased from 1.5 to 3 years, remaining stable to 4.5 years. This longitudinal pattern was related to higher Processing Speed (WPPSI-IV) scores at 4.5 years. Children born ELGA displayed higher AUCg than VLGA, particularly at age 3, driven by higher Pre-test cortisol levels. Overall, relative to those born VLGA, children born ELGA displayed greater cortisol responsivity to cognitive challenge. A higher setpoint of cortisol levels at age 3-years in children born ELGA may reflect altered HPA axis regulation more broadly and may contribute to difficulties with information processing in this population, critical for academic and social success.

Methylthioadenosine Phosphorylase Genomic Loss in Advanced Gastrointestinal Cancers.

BACKGROUND: One of the most common sporadic homozygous deletions in cancers is 9p21 loss, which includes the genes methylthioadenosine phosphorylase (MTAP), CDKN2A, and CDKN2B, and has been correlated with worsened outcomes and immunotherapy resistance. MTAP-loss is a developing drug target through synthetic lethality with MAT2A and PMRT5 inhibitors. The purpose of this study is to investigate the prevalence and genomic landscape of MTAP-loss in advanced gastrointestinal (GI) tumors and investigate its role as a prognostic biomarker. MATERIALS AND METHODS: We performed next-generation sequencing and comparative genomic and clinical analysis on an extensive cohort of 64 860 tumors comprising 5 GI cancers. We compared the clinical outcomes of patients with GI cancer harboring MTAP-loss and MTAP-intact tumors in a retrospective study. RESULTS: The prevalence of MTAP-loss in GI cancers is 8.30%. MTAP-loss was most prevalent in pancreatic ductal adenocarcinoma (PDAC) at 21.7% and least in colorectal carcinoma (CRC) at 1.1%. MTAP-loss tumors were more prevalent in East Asian patients with PDAC (4.4% vs 3.2%, P = .005) or intrahepatic cholangiocarcinoma (IHCC; 6.4% vs 4.3%, P = .036). Significant differences in the prevalence of potentially targetable genomic alterations (ATM, BRAF, BRCA2, ERBB2, IDH1, PIK3CA, and PTEN) were observed in MTAP-loss tumors and varied according to tumor type. MTAP-loss PDAC, IHCC, and CRC had a lower prevalence of microsatellite instability or elevated tumor mutational burden. Positive PD-L1 tumor cell expression was less frequent among MTAP-loss versus MTAP-intact IHCC tumors (23.2% vs 31.2%, P = .017). CONCLUSION: In GI cancers, MTAP-loss occurs as part of 9p21 loss and has an overall prevalence of 8%. MTAP-loss occurs in 22% of PDAC, 15% of IHCC, 8.7% of gastroesophageal adenocarcinoma, 2.4% of hepatocellular carcinoma, and 1.1% of CRC and is not mutually exclusive with other targetable mutations.

Updates in the Management of Congenital Melanocytic Nevi.

Congenital melanocytic nevi (CMN) carry an increased risk of melanoma and may be disfiguring, and consensus regarding treatment recommendations is lacking. While clinical monitoring is the standard of care, many caregivers are interested in its removal to prevent psychosocial burden or to decrease risk. Although melanoma can occur regardless of CMN removal, there are a variety of treatments that may offer improved cosmesis or local symptom control, including surgical excision, laser therapy, and other superficially destructive techniques. Regardless of the selected management, these patients are monitored for ongoing melanoma risk. An extensive discussion with families regarding the risks and benefits of observation versus active intervention is essential. To facilitate these discussions, we herein summarize current CMN management strategies and considerations.

Somatic and Depressive Symptoms Among Children From Latin America and the English-Speaking Caribbean.

BACKGROUND: The extent to which depression is associated with somatic complaints in children from the English-speaking Caribbean and Latin America is not well established. OBJECTIVE: We sought to explore the association between depressive and somatic symptoms among children from the English-speaking Caribbean and Latin America, while accounting for age, sex, socioeconomic status, cultural background, and anxiety score. METHOD: 1541 elementary school children, ages 9-12 years, from the English-speaking Caribbean and Latin America completed the Adolescent Depression Rating Scale (ARDS), the Numeric 0-10 Anxiety Self-Report Scale and the Children's Somatic Symptom Inventory-24 (CSSI-24). T-tests and ANOVA's were used to compare CSSI-24 and ARDS scores among countries, and the CSSI-24 scores of children with (ARDS ≥ 4) and without likely clinically significant depression. Regression analyses assessed possible predictors of CSSI-24 score. RESULTS: Depressive and somatic symptom scores were highest among the Jamaican children and lowest among the Colombian children (p < .001). Children with likely clinically significant depression exhibited higher mean somatic symptom scores (p < .001). Depressive symptom scores predicted somatic symptom scores (p < .001). CONCLUSIONS: Depressive symptoms were a strong predictor of reporting somatic symptoms. Knowledge of this association may facilitate better recognition of depression among youth.

Non-vascular intracranial lesions in three children with PHACE association.

PHACE (posterior fossa malformations, hemangiomas, arterial anomalies, cardiac anomalies, eye anomalies) association has many recognized clinical features. A link between PHACE and non-vascular intracranial lesions has not been well-described. We report three pediatric patients with PHACE and non-vascular intracranial lesions.

Age 70 +/- 5 Years and Cancer-Specific Outcomes After Treatment of Localized Prostate Cancer: A Systematic Review.

A secondary analysis of 2 randomized Radiation Therapy Oncology Group trials demonstrated that age ≥70 years was a favorable prognostic factor among men treated with external beam radiation therapy (EBRT). In contrast, several series based on men undergoing radical prostatectomy (RP) suggested that older age was an unfavorable prognostic factor. Our study was initiated to determine whether these observations reflect a true but paradoxical underlying age-related treatment-dependent biological phenomenon. We conducted a systematic review (PubMed, January 1, 1999-January 30, 2023) evaluating the effect of age on cancer-specific outcomes after definitive local treatment with either RP or EBRT. Our main objective was to assess possible interactions between age (using a cutoff of 70 +/- 5 years) and treatment type, with regard to adverse cancer-specific outcomes (eg, pathology, biochemical failure, distant metastasis, or prostate cancer-specific survival). Forty-five studies were selected for inclusion in this systematic review, including 30 and 15 studies with patients treated with RP and EBRT, respectively. Among patients treated with RP, 10 (50%) of these studies suggested that older age was associated with worse outcome(s) after RP. None suggested that age was a favorable prognostic factor after RP. Among the EBRT-based studies, 8 (53%) suggested that older age was associated with better outcomes, with an additional 3 studies (21%) trending to support a better outcome. None of these studies involving EBRT suggested that older age was an adverse prognostic factor. This systematic review suggests that age using a categorical cutoff of 70 +/- 5 years may be an adverse prognostic factor for men undergoing RP but a favorable prognostic factor for men treated with EBRT. Further research is needed to validate these findings.

Features, management, and outcomes of pediatric scalp melanomas.

Pediatric melanoma of the scalp has the highest mortality of any anatomic location. We describe five pediatric patients with a diagnosis of scalp melanoma receiving care at Massachusetts General Hospital and/or Boston Children's Hospital from 2018 through 2022. Melanoma presented in diverse contexts: cellular blue nevus-associated, compound nevus-associated, spitzoid, nodular, and superficial spreading subtypes. This study describes a range of melanoma presentations and emphasizes the need for additional compilation of data on pediatric scalp melanomas to promote their recognition and improve patient care.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Association of maternal education, neighborhood deprivation, and racial segregation with gestational age at birth by maternal race/ethnicity and United States Census region in the ECHO cohorts.

Background: In the United States, disparities in gestational age at birth by maternal race, ethnicity, and geography are theorized to be related, in part, to differences in individual- and neighborhood-level socioeconomic status (SES). Yet, few studies have examined their combined effects or whether associations vary by maternal race and ethnicity and United States Census region. Methods: We assembled data from 34 cohorts in the Environmental influences on Child Health Outcomes (ECHO) program representing 10,304 participants who delivered a liveborn, singleton infant from 2000 through 2019. We investigated the combined associations of maternal education level, neighborhood deprivation index (NDI), and Index of Concentration at the Extremes for racial residential segregation (ICERace) on gestational weeks at birth using linear regression and on gestational age at birth categories (preterm, early term, post-late term relative to full term) using multinomial logistic regression. Results: After adjustment for NDI and ICERace, gestational weeks at birth was significantly lower among those with a high school diploma or less (-0.31 weeks, 95% CI: -0.44, -0.18), and some college (-0.30 weeks, 95% CI: -0.42, -0.18) relative to a master's degree or higher. Those with a high school diploma or less also had an increased odds of preterm (aOR 1.59, 95% CI: 1.20, 2.10) and early term birth (aOR 1.26, 95% CI: 1.05, 1.51). In adjusted models, NDI quartile and ICERace quartile were not associated with gestational weeks at birth. However, higher NDI quartile (most deprived) associated with an increased odds of early term and late term birth, and lower ICERace quartile (least racially privileged) associated with a decreased odds of late or post-term birth. When stratifying by region, gestational weeks at birth was lower among those with a high school education or less and some college only among those living in the Northeast or Midwest. When stratifying by race and ethnicity, gestational weeks at birth was lower among those with a high school education or less only for the non-Hispanic White category. Conclusion: In this study, maternal education was consistently associated with shorter duration of pregnancy and increased odds of preterm birth, including in models adjusted for NDI and ICERace.

Retrospective Data Analysis of the Use of an Autologous Multilayered Leukocyte, Platelet, and Fibrin Patch for Diabetic Foot Ulcers Treatment in Daily Clinical Practice.

OBJECTIVE: To describe the healing outcome of chronic, hard-to-heal diabetic foot ulcers (DFUs) treated with an autologous multilayered leukocyte, platelet, and fibrin (MLPF) patch in addition to the best standard of care, in a real-world clinical setting of two US amputation preventive centers. METHODS: In this retrospective study of patients treated between September 2021 and October 2022, the authors analyzed DFU healing outcomes based on Wound, Ischemia, and foot Infection-derived amputation risk. RESULTS: All 36 patients had a diagnosis of type 2 diabetes and 29 (81%) were male. Their average age was 61.4 years, body mass index was 29.2 kg/m2, and glycated hemoglobin was 7.9. Twenty-seven patients (78%) were diagnosed with peripheral vascular disease, 20 (56%) underwent a peripheral vascular procedure, 15 (42%) had a prior amputation, and 6 (17%) were on hemodialysis. Average wound size was 4.9 cm2, and wound age was 9.5 months. Twelve patients (32%) were classified as low risk, 15 (39%) as moderate risk, and 11 (29%) as high risk for amputation. Within 12 weeks of the first MLPF patch application, nine wounds (24%) healed. After 20 weeks, 23 wounds (61%) were closed, and by follow-up, 30 wounds (79%) healed. No amputations were noted. Compared with published data, 40% fewer patients underwent readmission within 30 days, with 72% shorter admission duration. CONCLUSIONS: Real-world clinical experiences using the MLPF patch to treat hard-to-heal DFUs resulted in the majority of wounds healing. Few patients experienced a readmission within 30 days, and the average admission duration was short.

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE.

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key regulators in cellular immunity. However, their dense O-glycosylation remains enigmatic, primarily due to the challenges associated with studying mucin domains. Here, we demonstrate that the mucinase SmE has a unique ability to cleave at residues bearing very complex glycans. SmE enables improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we perform molecular dynamics (MD) simulations of TIM-3 and -4 to understand how glycosylation affects structural features of these proteins. Finally, we use these models to investigate the functional relevance of glycosylation for TIM-3 function and ligand binding. Overall, we present a powerful workflow to better understand the detailed molecular structures and functions of the mucinome.

Cortisol levels are related to neonatal pain exposure in children born very preterm at age 18 months in two independent cohorts.

Exposure to pain-related stress from frequent invasive procedures in the neonatal intensive care unit (NICU) has been associated with altered physiological stress regulation, neurodevelopment, and behavior in children born very preterm (≤32 weeks gestation). Previously, in a cohort born 2003-2006 (Cohort 1), we found that, at 18 months corrected age (CA), children born extremely low gestational age (ELGA; 24-28 weeks) and very low gestational age (VLGA; 29-32 weeks), had higher pre-test cortisol levels and a different pattern of cortisol output across a developmental assessment involving cognitive challenge compared to children born full-term (FT; 39-41 weeks). Also, greater neonatal pain-related stress exposure among the preterm children was related to higher pre-test cortisol levels. Given the adverse long-term effects of neonatal pain in preterm infants and the ensuing rise in clinical concerns to appropriately manage pain in the NICU in recent years, we aimed to examine whether our findings from Cohort 1 would still be evident in an independent cohort (Cohort 2) born 2006-2011 and recruited from the same tertiary NICU in Vancouver, Canada. We also compared the cortisol patterns, clinical and socio-demographic factors, and their interrelationships between the two cohorts. In Cohort 2, our findings using multi-level modeling support and extend our earlier findings in Cohort 1, demonstrating that children born ELGA display higher pre-test cortisol levels than FT. As well, greater cortisol output across assessment was related to more anxiety/depressive behaviors in children born VLGA. Importantly, children born ELGA were exposed to less neonatal pain/stress, mechanical ventilation, and morphine in Cohort 2 than Cohort 1. In both cohorts, however, cortisol levels and patterns were related to neonatal pain/stress and clinical factors (days on mechanical ventilation, overall morphine exposure). Despite less exposure to pain/stress and adverse clinical factors in Cohort 2 compared to Cohort 1, cortisol levels and patterns across cognitive challenge in preterm children at 18-month CA were consistent across the two independent cohorts. These findings highlight that, despite improvements to neonatal care, children born extremely preterm continue to display altered HPA axis activity, which is associated with their poorer neurodevelopmental and behavioral outcomes.