PLoS Med Research Articles

P-1092. Re-analysis of Pivmecillinam Randomized Clinical Trial Data According to 2019 US Food and Drug Administration Guidance for the Treatment of Uncomplicated Urinary Tract Infection

Abstract Background Due to increasing prevalence of resistant uropathogens, there is urgent need in the USA for effective new oral antibiotics for treatment of uncomplicated urinary tract infection (uUTI). Pivmecillinam has been used to treat uUTI in Canada and Europe for > 40 years, is recommended as a first-line agent by the Infectious Diseases Society of America, and has high microbiologic activity against most antibiotic-resistant Enterobacterales. We present a re-analysis of clinical trial data, as per 2019 US Food and Drug Administration (FDA) guidance, that supported the recent US approval of pivmecillinam for treatment of uUTI.Table 1.Composite response rates at test-of-cure (micro-ITT population)185 mg of pivmecillinam is equivalent to 200 mg of pivmecillinam hydrochloride.CI, confidence interval; micro-ITT, microbiological intent-to-treat; QID, four times daily; TID, three times daily.1. Ferry SA et al. Scand J Prim Health Care. 2007;25:49–57. 2. Menday AP. Intl J Antimicrob Agents. 2000;13:183–187. 3. Vik I et al. PLoS Med. 2018;15:e1002569. Methods Detailed subject-level clinical data were retrieved from three historic randomized controlled trials (RCTs) including subjects treated with pivmecillinam 185 mg, three times daily for 3–7 days, and re-analyzed in accordance with the 2019 FDA guidance for uUTI. Efficacy endpoints were composite (clinical and microbiologic) response rates, clinical response rates, and microbiologic response rates, analyzed in the microbiological intent-to-treat population (urine culture ≥ 105 colony-forming units/mL; ≤ 2 microorganism species; no baseline pathogen non-susceptible to active comparator).Table 2.Clinical response rates at test-of-cure (micro-ITT population)185 mg of pivmecillinam is equivalent to 200 mg of pivmecillinam hydrochloride.CI, confidence interval; micro-ITT, microbiological intent-to-treat; QID, four times daily; TID, three times daily. 1. Ferry SA et al. Scand J Prim Health Care. 2007;25:49–57. 2. Menday AP. Intl J Antimicrob Agents. 2000;13:183–187. 3. Vik I et al. PLoS Med. 2018;15:e1002569. Results Efficacy in uUTI was demonstrated for the recommended dosage regimen of pivmecillinam in the three studies (total N=369 pivmecillinam, N=385 comparators). Composite response rates for pivmecillinam ranged from 62.0% to 71.7% (Table 1), clinical response rates from 63.5% to 82.7% (Table 2), and microbiologic response rates from 74.3% to 86.9% (Table 3). Efficacy of pivmecillinam was superior to placebo or ibuprofen and similar to cephalexin. Clinical and microbiologic response rates for pivmecillinam and comparators were generally numerically lower in the re-analysis versus original reports, reflecting the more stringent 2019 FDA criteria.Table 3.Microbiologic response rates at test-of-cure (micro-ITT population)185 mg of pivmecillinam is equivalent to 200 mg of pivmecillinam hydrochloride.CI, confidence interval; micro-ITT, microbiological intent-to-treat; QID, four times daily; TID, three times daily. 1. Ferry SA et al. Scand J Prim Health Care. 2007;25:49–57. 2. Menday AP. Intl J Antimicrob Agents. 2000;13:183–187. 3. Vik I et al. PLoS Med. 2018;15:e1002569. Conclusion This re-analysis of RCT data confirmed the efficacy of pivmecillinam in uUTI, and was used to support the recent US approval of oral pivmecillinam, 185 mg three times daily for 3–7 days, for the treatment of female patients aged ≥ 18 years with uUTI caused by susceptible isolates of Escherichia coli, Proteus mirabilis, and Staphylococcus saprophyticus. Disclosures Keith S. Kaye, MD, MPH, Allecra: Advisor/Consultant|CARB-X: Advisor/Consultant|GSK: Advisor/Consultant|Merck: Advisor/Consultant|Shionogi: Advisor/Consultant|Spero: Advisor/Consultant Anita F. Das, PhD, Cidara: Advisor/Consultant|Contrafect: Advisor/Consultant|Iterum Therapeutics: Advisor/Consultant|Paratek: Advisor/Consultant|Utility therapeutics: Advisor/Consultant Niels Frimodt-Møller, MD DMSc, Eli Lilly Ltd: Stocks/Bonds (Private Company)|Pfizer Ltd: Stocks/Bonds (Private Company)|Rosco A/S: Advisor/Consultant Kalpana Gupta, MD, GlaxoSmithKline: Advisor/Consultant|IDSA GL on UTI: Uncompensated author|Iterum Therapeutics: Advisor/Consultant|PhenUtest Diagnostics: Advisor/Consultant|Qiagen Inc.,: Advisor/Consultant|UpToDate: Royalties for UTI topics|Utility Therapeutics Ltd: Advisor/Consultant Thomas Lodise, Jr., Pharm.D., PhD, MERCK: Advisor/Consultant Anne Santerre Henriksen, PhD, Utility therapeutics: Advisor/Consultant Morton Alexander, PhD, SNIPR biome: Shareholder|SNIPR biome: Stocks/Bonds (Private Company)|Union therapeutics: Shareholder|Union therapeutics: Stocks/Bonds (Private Company)|Utility therapeutics: Board Member|Utility therapeutics: Ownership Interest Florian Wagenlehner, MD, Astellas: Advisor/Consultant|AstraZeneca: Advisor/Consultant|Bionorica: Advisor/Consultant|DFG (German Research Foundation) funded research group BARICADE (FOR5427/1-466687329): Speaker|GSK: Advisor/Consultant|GSK: Principal investigator in a GSK-sponsored study|Janssen: Advisor/Consultant|Klosterfrau: Advisor/Consultant|MIP Pharma: Advisor/Consultant|OM Pharma: Advisor/Consultant|Spero: Advisor/Consultant|VenatoRX: Advisor/Consultant

12500 Objective Evaluation Of Clustering Methods For Resolving The Heterogeneity Of PCOS

Abstract Disclosure: K. Brewer: None. R. Sisk: None. M. Dapas: None. C. Li: None. A. Dunaif: Consulting Fee; Self; AcaciaBio, Inc, Neurocine Biosciences, Inc. Speaker; Self; Quest Diagnostics. Other; Self; Co-Editor Endocrine Today, Healio, Slack Inc. Clustering methods have been used to resolve heterogeneity within complex diseases to elucidate underlying biologic mechanisms. We (Dapas et al. PLoS Med, 2020) and others have applied these methods to PCOS and identified discrete subtypes, including those that capture distinct reproductive or metabolic features. We objectively compared two widely used methods, hierarchical clustering (HC), which recursively merges subjects based on their similarity, and k-means (Km), which iteratively groups individuals to minimize their distance to cluster centroids. The number of clusters (k) was predefined in both approaches. We compared HC vs Km in 874 European ancestry PCOS cases diagnosed by NIH criteria using 8 traits (BMI, testosterone, SHBG, DHEAS, LH, FSH, fasting insulin, fasting glucose) and 9 traits with the addition of AMH. The ConsensusClusterPlus R package was used to evaluate cluster stability of both approaches with k=2, 3, or 4, using 8 or 9 traits. Genomewide association study (GWAS) meta-analysis was performed as reported with a discovery cohort (620 cases, 2951 controls) and a replication cohort (371 cases, 926 controls), except that genotypes were imputed to the newer TOPMED (r2) panel. Clustering using 8 traits and k=3 resulted in the best cluster stability for both HC and Km (pairwise consensus proportion: HC 0.98, Km 0.93) compared to k=2 with 8 traits (HC 0.88, Km 0.88) or 9 traits (HC 0.95, Km 0.92). No stability was observed for k=4 with 8 or 9 traits using either method (range: 0.35-0.66). Km showed slightly better cluster separation than HC (average silhouette width: Km 0.12 vs HC 0.09) with k=3 and 8 traits. The biologic relevance of the three PCOS subtypes, which we have designated as reproductive (cases with higher LH, FSH, SHBG), metabolic (higher BMI, insulin, glucose), and background, was assessed by GWAS, an orthogonal (i.e., uncorrelated) approach for subtype confirmation. The subtypes identified with HC had genomewide significant associations (metabolic subtype, c9orf3/FANCC, rs10761370, minor allele frequency [MAF] 0.47, p=1.21 x 10-8; background subtype, FSHB/ARL14EP, rs10835649, MAF 0.17, p=8.49 x 10-[1]0). There were no genomewide significant signals when the subtypes were identified with Km despite having a better cluster separation. In summary, only HC clusters appeared to capture biologically meaningful differences since two of the three subtypes thus identified were associated with genomewide significant loci. Neither the addition of AMH nor increasing the number of groups improved clustering metrics. Our results emphasize the importance of independent validation of clustering approaches using an orthogonal confirmation strategy such as GWAS. We conclude that HC clustering using 8 traits is superior to Km for resolving the genetic heterogeneity of PCOS. Presentation: 6/2/2024

12500 Objective Evaluation Of Clustering Methods For Resolving The Heterogeneity Of PCOS

Abstract Disclosure: K. Brewer: None. R. Sisk: None. M. Dapas: None. C. Li: None. A. Dunaif: Consulting Fee; Self; AcaciaBio, Inc, Neurocine Biosciences, Inc. Speaker; Self; Quest Diagnostics. Other; Self; Co-Editor Endocrine Today, Healio, Slack Inc. Clustering methods have been used to resolve heterogeneity within complex diseases to elucidate underlying biologic mechanisms. We (Dapas et al. PLoS Med, 2020) and others have applied these methods to PCOS and identified discrete subtypes, including those that capture distinct reproductive or metabolic features. We objectively compared two widely used methods, hierarchical clustering (HC), which recursively merges subjects based on their similarity, and k-means (Km), which iteratively groups individuals to minimize their distance to cluster centroids. The number of clusters (k) was predefined in both approaches. We compared HC vs Km in 874 European ancestry PCOS cases diagnosed by NIH criteria using 8 traits (BMI, testosterone, SHBG, DHEAS, LH, FSH, fasting insulin, fasting glucose) and 9 traits with the addition of AMH. The ConsensusClusterPlus R package was used to evaluate cluster stability of both approaches with k=2, 3, or 4, using 8 or 9 traits. Genomewide association study (GWAS) meta-analysis was performed as reported with a discovery cohort (620 cases, 2951 controls) and a replication cohort (371 cases, 926 controls), except that genotypes were imputed to the newer TOPMED (r2) panel. Clustering using 8 traits and k=3 resulted in the best cluster stability for both HC and Km (pairwise consensus proportion: HC 0.98, Km 0.93) compared to k=2 with 8 traits (HC 0.88, Km 0.88) or 9 traits (HC 0.95, Km 0.92). No stability was observed for k=4 with 8 or 9 traits using either method (range: 0.35-0.66). Km showed slightly better cluster separation than HC (average silhouette width: Km 0.12 vs HC 0.09) with k=3 and 8 traits. The biologic relevance of the three PCOS subtypes, which we have designated as reproductive (cases with higher LH, FSH, SHBG), metabolic (higher BMI, insulin, glucose), and background, was assessed by GWAS, an orthogonal (i.e., uncorrelated) approach for subtype confirmation. The subtypes identified with HC had genomewide significant associations (metabolic subtype, c9orf3/FANCC, rs10761370, minor allele frequency [MAF] 0.47, p=1.21 x 10-8; background subtype, FSHB/ARL14EP, rs10835649, MAF 0.17, p=8.49 x 10-[1]0). There were no genomewide significant signals when the subtypes were identified with Km despite having a better cluster separation. In summary, only HC clusters appeared to capture biologically meaningful differences since two of the three subtypes thus identified were associated with genomewide significant loci. Neither the addition of AMH nor increasing the number of groups improved clustering metrics. Our results emphasize the importance of independent validation of clustering approaches using an orthogonal confirmation strategy such as GWAS. We conclude that HC clustering using 8 traits is superior to Km for resolving the genetic heterogeneity of PCOS. Presentation: 6/2/2024

Abstract 4867: Longitudinal latent class analysis to further understand trajectory of density over time and risk of breast cancer

Abstract Background: It is clinically important to refine strategies to manage women with dense breasts as they represent approximately 50% of all women screened. Change in breast density is related to risk of breast cancer.1 We also must understand this change in density over time in women with dense and non-dense breasts. While population level data have been reported to estimate decrease in density with age, this has largely used digitized film images.2 Expanding use of digital mammography and repeated screening generates a large library of images for each woman, offering the potential to use more of the image data. Methods: We use longitudinal latent class analysis to cluster women and estimate their change in density over time. To evaluate the groups of women with different patterns of change in density we fit latent class models to our previously published data.1 This includes 289 pathology confirmed cases of breast cancer and 658 controls among women followed from November 3, 2008 to October 31, 2020. Results: Among women with dense breasts, there are 107 who developed breast cancer during follow-up and 267 control women who remained free from breast cancer. In the women without dense breasts, 173 cases and 383 controls were evaluated and showed similar age and BMI within the controls as seen in women with dense breasts. Prevalence of family history and history of benign breast biopsy did not differ between the two groups. We observe this latent class and trajectory of density phenomenal separately for dense and non-dense groups in our data. We show that the data define 2 classes, those who have decrease in density over time and those who have increase over time. The odds ratio (OR) for cancer comparing decline vs. incline is 4.9 in women with dense breasts (BI-RADS C, D) and 6.17 on women with non-dense breasts (BI-RADS A, B). Discussion: To refine our understanding of patterns of change in breast density over time in relation to breast cancer risk we fit latent class models. Longitudinal change matters as reflected in the 2-class model. This is independent of starting density and confirms that a one-time measure of density is not enough to define level of risk for subsequent breast cancer. Further work is needed to better define the drivers and inhibitors of decline in breast density over time given this change over time as women age is a universal phenomenon.2 1. Jiang S, Bennett DL, Rosner BA, Colditz GA. Longitudinal Analysis of Change in Mammographic Density in Each Breast and Its Association With Breast Cancer Risk. JAMA Oncol 2023;9(6):808-814. (In English). DOI: 10.1001/jamaoncol.2023.0434. 2. Burton A, Maskarinec G, Perez-Gomez B, et al. Mammographic density and ageing: A collaborative pooled analysis of cross-sectional data from 22 countries worldwide. PLoS Med 2017;14(6):e1002335. DOI: 10.1371/journal.pmed.1002335. Citation Format: Graham A. Colditz, Debbie L. Bennett, Shu Jiang. Longitudinal latent class analysis to further understand trajectory of density over time and risk of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4867.

Convalescent Plasma and the US Expanded Access Program: A Personal Narrative.

Between early April 2020 and late August 2020, nearly 100,000 patients hospitalized with SARS-CoV2 infections were treated with COVID-19 convalescent plasma (CCP) in the US under the auspices of an FDA-authorized Expanded Access Program (EAP) housed at the Mayo Clinic. Clinicians wishing to provide CCP to their patients during that 5-month period early in the COVID pandemic had to register their patients and provide clinical information to the EAP program. This program was utilized by some 2,200 US hospitals located in every state ranging from academic medical centers to small rural hospitals and facilitated the treatment of an ethnically and socio-economically diverse cross section of patients. Within 6 weeks of program initiation, the first signals of safety were found in 5,000 recipients of CCP, supported by a later analysis of 20,000 recipients (Joyner et al. in J Clin Invest 130:4791-4797, 2020a; Joyner et al. in Mayo Clin Proc 95:1888-1897, 2020b). By mid-summer of 2020, strong evidence was produced showing that high-titer CCP given early in the course of hospitalization could lower mortality by as much as a third (Joyner et al. in N Engl J Med 384:1015-1027, 2021; Senefeld et al. in PLoS Med 18, 2021a). These data were used by the FDA in its August decision to grant Emergency Use Authorization for CCP use in hospitals. This chapter provides a personal narrative by the principal investigator of the EAP that describes the events leading up to the program, some of its key outcomes, and some lessons learned that may be applicable to the next pandemic. This vast effort was a complete team response to a crisis and included an exceptional level of collaboration both inside and outside of the Mayo Clinic. Writing just 4 years after the initiation of the EAP, this intense professional effort, comprising many moving parts, remains hard to completely understand or fully explain in this brief narrative. As Nelson Mandela said of the perception of time during his decades in prison, "the days seemed like years, and the years seemed like days."

Effect of the Oral LSD1 Inhibitor Ory-1001 on F-Retics in Baboons ( Papio anubis)

Elevated levels of Fetal Hemoglobin (HbF) reduce the severity of symptoms and increase life span in patients with sickle cell disease (SCD). Experiments performed in baboons ( P. anubis) have been instrumental in discovering the potent pharmocological agents that increase HbF in vivo. Experimental baboon studies have been directly translated to clinical trials in patients with sickle cell disease. Drugs such as DNMT1 and LSD1 inhibitors that attack the repressive epigenetic regulatory mechanisms that silence expression of the γ-globin gene in adult erythroid cells increase HbF in baboons to levels necessary to provide therapeutic relief in SCD patients. Dose-dependent adverse events associated with the use of these drugs are minimized by modifications of dose and schedule as shown by a clinical trial of oral THU-decitabine in 25 SCD patients (Molokie et al, Plos Med 2017;14(9):e10022382). Our recent results have shown that subcutaneous administration of the DNMT1 inhibitor decitabine and the LSD1 inhibitor RN-1 2d/week produced synergistic increases in F-retics, F cells and γ-globin mRNA in normal baboons and increased HbF to therapeutic levels in phlebotomized anemic baboons after administration of only four doses (Ibanez et al, Blood Adv 2023;7(15):3891-3902). The goal of this current study is to develop an oral combinatorial drug regimen using THU-decitabine and the LSD1 inhibitor ORY-1001 that could be directly translated to clinical trials. Our previous studies demonstrated the effectiveness of oral THU-decitabine to increase HbF levels in baboons (Lavelle et al, Blood 2012;119(5):1240-1247). In this study we have performed 16 dose-response experiments in baboons to test the effect of oral ORY-1001 at doses ranging between 8 and 24µg/kg/d. ORY-1001 was chosen because it exhibited a good safety profile with known pharmacokinetic and pharmacodynamic parameters in a recent study in patients with relapsed/refractory acute myeloid leukemia (Salamero et al, J Clin Oncol 2020; 38(36):4260-4273). Four doses were tested (24µg/kg, n=2; 16µg/kg n=4; 12µg/kg, n-=7; 8µg/kg, n=3). The drug was administered on a single day to facilitate further combinatorial studies with THU-decitabine. Preliminary data from a current clinical trial of THU-decitabine in SCD patients (NCT04055818) suggests that administering the drug on a 1d per week schedule effectively increased HbF levels in patients. ORY-1001 was administered by gavage on a single day (d1) and the effect on F-retics measured by flow cytometry in peripheral blood samples obtained daily between days 6 and 10. The peak F-retic response was observed on d8. A linear dose-response relationship on F-retics was observed (Figure 1, r=0.79). Complete blood counts (CBC) were also performed daily between d6 and d10. Adverse hematological effects were not observed with no incidences of neutropenia or thrombocytopenia (Table 1). No consistent effects on reticulocyte counts were observed (Table 1). These results show that a single oral dose of ORY-1001 is sufficient to increase HBF in baboons without any adverse hematological effects and provide the necessary dose-response data to pursue further combinatorial studies with oral THU-decitabine

Translating an HPFH-Mechanism into Oral Small Molecule Therapy for Beta-Hemoglobinopathies: Clinical Proof-of-Principle

Introduction: Hereditary persistence of fetal hemoglobin (HPFH) down-modulates co-inherited sickle cell disease (SCD) severity, encouraging characterization of HPFH-mechanisms for interventional reprise. One HPFH mechanism is polymorphisms in DNA methyltransferase 1( DNMT1) that decrease DNMT1 protein stability (Gong et al, Blood 2021), agreeing with biochemical observations that DNMT1 mediates repression of fetal hemoglobin (HbF) genes by BCL11A. Here we describe translation of this HPFH-mechanism into oral therapy for SCD patients at high-risk for early mortality (Steinberg et al, JAMA 2004) despite standard-treatments. Methods and Results: The small molecule decitabine (Dec) contains a pyrimidine ring modification that binds and depletes DNMT1 and a natural deoxyribose that incorporates into DNA without terminating chain synthesis: minimum biologically effective doses of Dec, to deplete DNMT1 without cytotoxicity, can thus be found (reviewed in Zavras et al, Clin Ca Res, 2021). Other key properties of Dec are tropism to hematopoietic precursors, driven by high expression in these cells of deoxycytidine kinase that phosphorylates Dec, and rapid catabolism in vivo by cytidine deaminase (CDA), that severely restricts Dec bioavailability and half-life. We therefore combined Dec with a CDA-inhibitor tetrahydrouridine (THU): dose and schedule of THU/Dec that depleted DNMT1 without cytotoxicity to hematopoietic precursors in vivo were identified in non-human primate studies and Phase 1 clinical trials (Molokie et al, PLoS Med 2017), and here we describe interim results from proof-of-principle study in patients with SCD ( Table 1). Study interventions were oral THU/Dec (~10/0.2 mg/kg) 1X/week with or without oral nicotinamide (vitamin B3) 1 g/day. B3 was studied because it is a direct precursor of NADH, a fundamental antioxidant currency and also a cofactor for epigenetic enzymes that channel hematopoiesis to erythropoiesis vs megakaryopoiesis, to potentially counter an expected side-effect of THU/Dec of increasing platelets. Randomization was to THU/Dec vs B3 for 12 weeks, followed by THU/Dec + B3 for 12 weeks, then option to extend therapy for a further 24 weeks. Standard clinical and laboratory parameters were measured. Six patients enrolled, were randomized equally to THU/Dec or B3, and opted to extend. Dose-limiting toxicities (DLTs), including neutropenia or thrombocytosis, did not occur ( Table 1). In the year preceding study-treatment, 15 vaso-occlusive crises (VOC) requiring hospitalization were documented ( Table 1). By comparison, during 52 weeks on-study (48 weeks on-therapy, 4 weeks follow-up), the grade 3/4 adverse events were VOC hospitalizations x3 (1 during follow-up without therapy, 1 during B3 alone, 1 during B3 + THU/Dec therapy), hyperkalemia x2, and non-cardiac chest pain x1 ( Table 1)(p=0.04, paired t-test 2-sided). THU/Dec dose (7.6/0.15 mg/kg) was below the Phase 1 identified minimum biologically effective dose window (8-12/0.16-0.25 mg/kg, protocol since amended to accommodate high body weights) in one patient (TDN04) who accordingly did not have HbF% or hemoglobin increases ( Fig.1). In the 5 other patients, HbF increases (pre-treatment to maximum) were: 9.4 to 17.3, 5.9 to 11, 3.4 to 9.3, 3.3 to 16.3 and 5.7 to 10 % ( Fig.1). There were corresponding improvements in hemolysis and coagulation pathway parameters (LDH, total bilirubin, D-Dimer), and total hemoglobin increased from 9.9 to 11.5, 8.7 to 10.6, 6.6 to 7.7, 5.7 to 7.1 and 8 to 9.7 g/dL respectively. TDN01 and 06, the two patients with lowest pre-treatment hemoglobin, requested and resumed THU/Dec post-study by Expanded Access. Conclusions: This is the longest-term treatment to date of oral THU/Dec to target DNMT1 in patients with SCD, with or without B3. Subjective and objective improvements were sustained without DLTs in high-risk patients who displayed known negative prediction markers for response to hydroxyurea (HbF% <7.5%, reticulocyte count <300 x10 9/L - Steinberg et al, Blood 1997). The safety and efficacy signals align with scientific target validation data, and together with accessibility, support relevance to SCD patient needs and goals worldwide, bearing in mind major study limitations of single-site enrollment and only 6 patients.

OR20-02 Trans-Ethnic Analysis Of PCOS Subtype Genomewide Association Signals Reveals 3 Shared Subtype-Specific Loci

Abstract Disclosure: K. Brewer: None. R. Sisk: None. H. Lee: None. L.M. Moolhuijsen: None. K. van der Ham: None. Y. Louwers: None. J.A. Visser: None. M. Dapas: None. S. Franks: None. J.S. Laven: None. C. Li: None. A.E. Dunaif: None. The phenotypic variation observed in PCOS is suggestive of underlying genetic heterogeneity, but a recent meta-analysis of European ancestry (EA) PCOS cases found that the genetic architecture of PCOS defined by NIH and Rotterdam diagnostic criteria was similar, suggesting that these criteria do not identify biologically distinct disease phenotypes. Using Hierarchical Clustering (HC) in EA NIH PCOS cases, we have identified discrete, stable PCOS subtypes, which we called reproductive and metabolic (Dapas. PLoS Med, 2020); cases that did not cluster were designated as “background”. These subtypes appeared to capture biologically meaningful differences because they were associated with distinct genomewide significant loci. We were unable to replicate 4 of 6 loci since the lead SNPs were neither genotyped nor imputed on the array used for replication, the Metabochip custom array for cardiometabolic traits with additional PCOS-relevant SNPs. Here, we report an updated EA genomewide association study (GWAS) meta-analysis with additional EA NIH PCOS cases, and a trans-ethnic meta-analysis including a Korean ancestry (KA) NIH PCOS cohort (Lee. Hum Reprod, 2015). HC was applied to cases in both cohorts to identify PCOS subtypes. The EA discovery cohort, 620 cases and 2951 controls, was genotyped with the Illumina OmniExpress array. The replication EA cohort, 371 cases and 926 controls, was genotyped with the Metabochip. The KA cohort, 417 cases and 926 controls, was genotyped with the HumanOmni1-Quad v1 array. The imputation of previously reported genotype data was updated using the larger, more deeply sequenced TOPMed (R2) imputation panel. Nine loci were genomewide significant in the EA meta-analysis: 5 novel loci (reproductive subtype); 2 loci (metabolic subtype), including c9orf3; 2 loci (background subtype), including FSHB/ARL14P. Five of 6 other lead SNPs in the original analysis remained nominally significant. Three loci were significant in the trans-ethnic meta-analysis: a novel locus on chr 3, EPHA6, a receptor tyrosine kinase that binds promiscuously GPI-anchored ephrin-A family ligands (rs140766105, p=1.08 x 10-8, reproductive subtype); a previously identified GWAS locus on chr 9, c9orf3 (AOPEP), an aminopeptidase that catalyzes the hydrolysis of amino acid residues from the N-terminus of peptides (rs10761370, p=4.12 x 10-9, metabolic subtype); a previously identified GWAS locus on chr 11, FSHB/ARL14P, FSHB encodes the β-subunit of FSH and is associated with general fertility status, (rs10835649, 1.23 x 10-11, background subtype). We have replicated the 9 distinct subtype loci in the EA meta-analysis providing orthogonal (independent) validation suggesting these subtypes have distinct genetic architecture. For the first time, a trans-ethnic meta-analysis demonstrates that 3 subtype-specific loci play a role in PCOS pathogenesis across diverse populations. Presentation Date: Saturday, June 17, 2023

O-014 Clustering identifies distinct subtypes of PCOS – Towards a Rationale Approach to PCOS classification

Abstract Study question Does unsupervised clustering identify biologically distinct subtypes in a cohort of women with polycystic ovary syndrome (PCOS) diagnosed by Rotterdam criteria? Summary answer This study demonstrates that unsupervised hierarchical clustering of eight pre-defined quantitative reproductive and metabolic traits identifies biologically distinct subtypes in women with PCOS. What is known already PCOS is a common, heterogeneous, endocrine disorder in women of reproductive-age. PCOS diagnosed by NIH or non-NIH Rotterdam criteria or by self-report is generally genetically similar. Using Hierarchical Clustering (HC), we have previously identified discrete, stable PCOS subtypes, which we designated reproductive (higher LH, FSH, SHBG) and metabolic (higher BMI, insulin, glucose), in a United States cohort of ∼900 PCOS women diagnosed by NIH criteria (Dapas et al. PLoS Med, 2020). The cases that did not cluster were designated “background subtype”. The subtypes appeared to capture biologically meaningful differences because they were associated with distinct and novel genome-wide significant loci. Study design, size, duration In the current study, we applied HC to the same traits (BMI, LH, FSH, DHEAS, SHBG, testosterone, fasting insulin and fasting glucose). We then assessed whether additional traits differed between the subtypes thus identified: anti-Müllerian hormone (AMH), total follicle count, modified Ferriman-Gallwey score, estrogen, TSH, DHEA, cortisol, androstenedione, prolactin, LDL, HDL, triglycerides and cholesterol. Participants/materials, setting, methods Women of European ancestry, aged 13-45 years, n = 2502, with PCOS according to the Rotterdam criteria were included; n = 1067 also fulfilled NIH criteria. All quantitative traits were log-transformed to approximate a normal distribution. Z-scores were used to compare the differences between the three clusters using ANCOVA, corrected for age. Pair-wise comparison of the different clusters was performed using Fisher’s least significance difference method and adjusted for multiple testing. Main results and the role of chance We replicated discrete subtypes in this large cohort of women with PCOS defined by the Rotterdam criteria. There were 1026 cases in the metabolic subtype, 450 cases in the reproductive subtype and 1026 in the background subtype. Cases in the reproductive subtype had significantly (all P < 0.001) higher serum AMH levels, follicle counts and HDL levels compared to the metabolic and background subtypes. These findings suggest that the reproductive subtype captures affected women with the alterations in folliculogenesis characteristic of PCOS, without using PCOM to define this subtype. In contrast, the cases in the metabolic subtype had significantly (all P < 0.001) higher triglyceride and LDL levels compared to the other subtypes providing further evidence that this subtype identifies cases with cardiometabolic risk. Androstenedione and TSH levels were significantly increased in the metabolic subtype compared to the background subtype (P < 0.001) and to both subtypes (P = 0.004), respectively. Cortisol and prolactin levels did not differ among the three subtypes. All results did not differ when the analysis was limited to NIH PCOS cases. Overall, our findings suggest that these PCOS subtypes have different etiologies and clinical outcomes. Subtyping may enable precision medicine approaches to the management of what is currently classified as PCOS. Limitations, reasons for caution A limitation of the study is that we have not replicated these findings in an independent cohort. We have not used an orthogonal method, such as genome-wide association, to confirm that the subtypes capture biologically distinct groups. Wider implications of the findings Taken together with our previous studies suggesting that the genetic architecture of these subtypes differs, the current study implies that PCOS consists of several etiologically distinct disorders. Our findings provide an example of the power of modern disease classification based on objective biologic differences rather than expert opinion. Trial registration number Not applicable

Blood transfusion for children in sub-Saharan Africa: 200 years on

In 1818, James Blundell completed the first successful human blood transfusion. This life-saving medical intervention has revolutionised patient care globally in the past two centuries. Nevertheless, many challenges remain in implementing safe blood transfusion procedures, particularly in sub-Saharan Africa 1 Ala F Allain JP Bates I et al. External financial aid to blood transfusion services in sub-Saharan Africa: a need for reflection. PLoS Med. 2012; 9e1001309 Crossref PubMed Scopus (53) Google Scholar where demand predominantly centres on acute paediatric severe anaemia and haemorrhagic complications of pregnancy and trauma. Severe anaemia, when the haemoglobin concentration drops below 6 g/dL, is very common in children admitted to hospital in sub-Saharan Africa. Infectious diseases, nutritional deficiencies, and sickle cell anaemia are the major underlying causes of anaemia. Outcomes remain unsatisfactory, with high mortality and high hospital readmission rates within 6 months of initial admission. 2 Phiri KS Calis JC Faragher B et al. Long term outcome of severe anaemia in Malawian children. PLoS One. 2008; 3e2903 Crossref PubMed Scopus (75) Google Scholar World Blood Donor Day on June 14, 2023, calls for donors to “Give blood, give plasma, share life, share often”. Not only is this a day to celebrate the charitable contributions of blood donors, it is an important opportunity to raise awareness that every pint of blood donated results in critically important life-sustaining therapy, especially for children in sub-Saharan Africa.

The irony of drinking to health and happiness

In testing the relationship between alcohol consumption in young people and depression in young adulthood, in The Lancet Psychiatry, Gemma Hammerton and colleagues 1 Hammerton G Lewis G Heron J Fernandes G Hickman M Lewis G The association of alcohol dependence and consumption during adolescence with depression in young adulthood, in England: a prospective cohort study. Lancet Psychiatry. 2023; (published online June 1.)https://doi.org/10.1016/S2215-0366(23)00138-4 Google Scholar address a very important question. Depression causes an enormous public health burden. WHO identifies depression as affecting more than 4% of the world's population and causing substantial disability. 2 WHODepression and other common mental disorders: global health estimates. World Health Organization, Geneva2017 Google Scholar Understanding how modifiable risk factors, such as alcohol consumption, influence the onset of depression in adulthood is therefore a crucial question for epidemiology to tackle. The Avon Longitudinal Study of Parents and Children (ALSPAC) is a highly appropriate data resource to use to address this question. ALSPAC is well placed in the UK's catalogue of large birth cohort studies, sitting between the 1970 British Cohort Study and the Millennium Cohort Study, uniquely assessing the development of children born in the 1990s. I was involved in a systematic review of available evidence on the adult outcomes of late adolescent alcohol consumption, 3 McCambridge J McAlaney J Rowe R Adult consequences of late adolescent alcohol consumption: a systematic review of cohort studies. PLoS Med. 2011; 8e1000413 Crossref PubMed Scopus (313) Google Scholar which concluded that the area needed more high-quality prospective cohort studies, and Hammerton and colleagues' work strongly contributes to this need. The association of alcohol dependence and consumption during adolescence with depression in young adulthood, in England: a prospective cohort studyPsychosocial or behavioural interventions that reduce the risk of alcohol dependence during adolescence could contribute to preventing depression in young adulthood. Full-Text PDF Open Access

Integrating substance use and mental health services

People living with HIV have an increased prevalence and frequency of medical, psychiatric, and substance-use disorders that result in increased age-matched morbidity and mortality. 1 Altice FL Kamarulzaman A Soriano VV Schechter M Friedland GH Treatment of medical, psychiatric, and substance-use comorbidities in people infected with HIV who use drugs. Lancet. 2010; 376: 367-387 Summary Full Text Full Text PDF PubMed Scopus (350) Google Scholar Alcohol, tobacco, and other substance use often co-occur with depression, anxiety, and chronic pain. This constellation of alcohol, substance, and mood-related (CASM) conditions disproportionately affects people with HIV. 2 Bershteyn A Richard E Zhou Q et al. Potential health benefits of integrated screening strategies for alcohol, tobacco, other substance use, depression, anxiety, and chronic pain among people living with HIV in the USA. Lancet HIV. 2022; 10: e118-e125 Google Scholar Integration of HIV services with other health services has been proposed as an important strategy to boost the sustainability of the global HIV response and contribute to the goal of ending AIDS by 2030. 3 Bulstra CA Hontelez JAC Otto M et al. Integrating HIV services and other health services: a systematic review and meta-analysis. PLoS Med. 2021; 18e1003836 Crossref Scopus (8) Google Scholar In The Lancet HIV, Anna Bershteyn and colleagues 2 Bershteyn A Richard E Zhou Q et al. Potential health benefits of integrated screening strategies for alcohol, tobacco, other substance use, depression, anxiety, and chronic pain among people living with HIV in the USA. Lancet HIV. 2022; 10: e118-e125 Google Scholar showed that primary care providers of people with HIV in the USA should consider comprehensive diagnostic assessment of CASM if one or more conditions screen positive, a strategy that could increase life expectancy by nearly a year, exceeding life expectancy gains from benchmark preventative care recommendations. 2 Bershteyn A Richard E Zhou Q et al. Potential health benefits of integrated screening strategies for alcohol, tobacco, other substance use, depression, anxiety, and chronic pain among people living with HIV in the USA. Lancet HIV. 2022; 10: e118-e125 Google Scholar Potential health benefits of integrated screening strategies for alcohol, tobacco, other substance use, depression, anxiety, and chronic pain among people living with HIV in the USA: a mathematical modelling studyPrimary care providers for people with HIV should consider comprehensive diagnostic assessment of CASM conditions if one or more conditions screen positive. Full-Text PDF

Self-administered OPAT (S-OPAT) is a potential cost-effective equivalent option for uninsured patients requiring OPAT

A clinical decision report using: Bhavan KP, Brown LS, Haley RW. Self-Administered Outpatient Antimicrobial Infusion by Uninsured Patients Discharged from a Safety-Net Hospital: A Propensity-Score-Balanced Retrospective Cohort Study. PLoS Med. 2015;12(12): e1001922. https://doi.org/10.1371/journal.pmed.1001922 for a patient with fungal peritonitis requiring OPAT.

RF10 | PMON217 Replication of PCOS Reproductive and Metabolic Subtypes in Diverse Cohorts – Towards a Rationale Approach to PCOS Classification

Abstract PCOS diagnosed by NIH or non-NIH Rotterdam criteria or by self-report is generally genetically similar. Using Hierarchical Clustering (HC), we have previously identified discrete, stable PCOS subtypes, which we designated reproductive (higher LH, FSH, SHBG) and metabolic (higher BMI, insulin, glucose), in a United States (US) cohort of ∼900 PCOS diagnosed by NIH criteria (Dapas et al. PLoS Med, 2020). The subtypes appeared to capture biologically meaningful differences because they were associated with distinct and novel genome-wide significant loci. In this study, we tested the hypothesis that the subtypes are a common feature of PCOS in regionally and ethnically diverse cohorts. All PCOS cases were ages 13-45 years, at least 2-years postmenarche and fulfilled NIH diagnostic criteria: 1) US European ancestry (EA), 1449; 2) Netherlands (NL) EA, 1067; 3) Greece (GR) EA, 568; 4) South Korea (KR) East Asian ancestry, 418. BMI, testosterone, SHBG, DHEAS, LH, FSH, fasting insulin, and fasting glucose levels were used for clustering. Trait Z-scores were compared among the cohorts. HC was adjusted for age and assay method. Both median BMI and BMI categories (nonobese, overweight, obese) defined by ancestry-specific cut points for EA and Asians differed (US>NL>GR>KR, all P<0.01). The prevalence of Class 2 (35.0-39.9) and Class 3 (≥ 40.0) was highest in US (P<0.001). The remaining traits were similar between cohorts prior to HC. Reproductive and metabolic subtypes were present in the 4 cohorts with similar Jaccard coefficients indicating cluster stability. The percent of cases was: reproductive subtype US 26%; NL 19%; GR, 62%; KR 20%; metabolic subtype US 53%; NL 61%; GR 22%; KR 31%. These differences were significant, except for the reproductive subtype in NL and KR. There was a 3-fold higher prevalence of the reproductive subtype in GR. BMI could not completely account for the metabolic subtype differences since KR had a higher prevalence of the metabolic subtype than GR, despite being significantly less obese. Remaining cases in each cohort who did not have a distinct pattern of trait distribution were designated "indeterminate" (US 21%; NL 20%; GR 16%; KR 49%). Reproductive and metabolic subtypes of PCOS diagnosed by NIH criteria are present in both regionally (US, Northern and Southern Europe) and ethnically (European and East Asian) diverse cohorts. Despite similar trait values, except for BMI, the prevalence of these subtypes varied among cohorts. Compared to other European and East Asian ancestry cohorts, the GR cohort had a strikingly higher prevalence of the reproductive and lower prevalence of the metabolic subtype. The reasons for these differences are being investigated but may reflect population-specific genetic variation. Nevertheless, our findings suggest that clustering algorithms capture biologically discrete subtypes in diverse PCOS populations diagnosed by NIH criteria. Presentation: Saturday, June 11, 2022 1:54 p.m. - 1:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Estimating antimicrobial resistance burden in Europe—what are the next steps?

Estimating antimicrobial resistance burden in Europe—what are the next steps?

Establishing the worth of deprescribing inappropriate medications: are we there yet?

Establishing the worth of deprescribing inappropriate medications: are we there yet?

Neuropsychiatric sequelae of COVID-19: long-lasting, but not uniform

Neuropsychiatric sequelae of COVID-19: long-lasting, but not uniform

Evaluation of data imputation strategies in complex, deeply-phenotyped data sets: the case of the EU-AIMS Longitudinal European Autism Project

An increasing number of large-scale multi-modal research initiatives has been conducted in the typically developing population, e.g. Dev. Cogn. Neur. 32:43-54, 2018; PLoS Med. 12(3):e1001779, 2015; Elam and Van Essen, Enc. Comp. Neur., 2013, as well as in psychiatric cohorts, e.g. Trans. Psych. 10(1):100, 2020; Mol. Psych. 19:659–667, 2014; Mol. Aut. 8:24, 2017; Eur. Child and Adol. Psych. 24(3):265–281, 2015. Missing data is a common problem in such datasets due to the difficulty of assessing multiple measures on a large number of participants. The consequences of missing data accumulate when researchers aim to integrate relationships across multiple measures. Here we aim to evaluate different imputation strategies to fill in missing values in clinical data from a large (total N = 764) and deeply phenotyped (i.e. range of clinical and cognitive instruments administered) sample of N = 453 autistic individuals and N = 311 control individuals recruited as part of the EU-AIMS Longitudinal European Autism Project (LEAP) consortium. In particular, we consider a total of 160 clinical measures divided in 15 overlapping subsets of participants. We use two simple but common univariate strategies—mean and median imputation—as well as a Round Robin regression approach involving four independent multivariate regression models including Bayesian Ridge regression, as well as several non-linear models: Decision Trees (Extra Trees., and Nearest Neighbours regression. We evaluate the models using the traditional mean square error towards removed available data, and also consider the Kullback–Leibler divergence between the observed and the imputed distributions. We show that all of the multivariate approaches tested provide a substantial improvement compared to typical univariate approaches. Further, our analyses reveal that across all 15 data-subsets tested, an Extra Trees regression approach provided the best global results. This not only allows the selection of a unique model to impute missing data for the LEAP project and delivers a fixed set of imputed clinical data to be used by researchers working with the LEAP dataset in the future, but provides more general guidelines for data imputation in large scale epidemiological studies.

Deaths related to the combination of pregabalin and other drugs of abuse: About two cases

The number of deaths involving pregabalin has dramatically increased in recent years. In the majority of the cases described in France, pregabalin is associated with opioids, mainly methadone, as well as benzodiazepines and other psychoactive drugs, notably antidepressants and antipsychotics. Association with alcohol, cannabis and psychostimulants is also mentioned in the literature (Evoy KE et al. Drugs 2021;81:125–56). In this context, pregabalin concentrations are usually high, above therapeutic concentrations, in order to achieve euphoric effects, to enhance the effects of other substances or to improve sociability (Hofmann M & Besson M. Psychiatry Res 2021;35:114193). The authors present two cases of unusual deaths, a few days apart, for which toxicological analyses, showing low concentrations of pregabalin, buprenorphine, clonazepam and psychostimulants, are in favor of new patterns of use. Case 1: a 30-year-old man with a known addiction to pregabalin and cocaine, was seen by a witness snorting buprenorphine. He was found dead two hours later. Case 2: A young homeless man is found dead in the street in the early hours of the morning. No information is available on his identity and medical history. In both cases, the autopsy performed within 48 hours only show diffuse polyvisceral congestion with major cerebral and pulmonary oedema and the pathological examination did not allow to determine the cause of death. The usual toxicological samples, including femoral blood, with sodium fluoride as preservative, were collected and complete toxicological analyses were requested. Analyses were performed in femoral blood. Alcohols were measured by HS-GC-FID. Non-targeted screening and simultaneous quantification was performed by LC-HRMS. Specific detection of drugs of abuse (DOA) was done by LC-MS/MS. In case 1, analyses showed the presence of pregabalin (4900 ng/mL), buprenorphine (2.5 ng/mL), norbuprenorphine (2.5 ng/mL), 7-aminoclonazepam (14 ng/mL), diazepam (51 ng/mL), nordiazepam (137 ng/mL), oxazepam (7 ng/mL), cocaine (4 ng/mL), benzoylecgonine (177 ng/mL), ecgonine methylester (33 ng/mL) and THC-COOH (4 ng/mL). In case 2, results showed the presence of pregabalin (5700 ng/mL), buprenorphine (4.5 ng/mL), norbuprenorphine (0.5 ng/mL), 7-aminoclonazepam (23 ng/mL), MDMA (1140 ng/mL) and MDA (54 ng/mL). Alcohol testing was negative in both cases. In both cases, the concentrations of pregabalin, benzodiazepines and buprenorphine are far below the toxic concentrations. While the risk of death when buprenorphine is combined with benzodiazepines at therapeutic doses, particularly when buprenorphine is injected or absorbed nasally (Sansone RA & Sansone LA. Innov clin NeuroSci 2015;12:32–6), is well described in the literature, the potential role of pregabalin remains to be determined. Indeed, if the combination of gabapentinoids/opioids/other depressants is known to favor the occurrence of respiratory depression, it seems that the favoring factors are a high dose, an advanced age and co-morbidities (chronic pulmonary disease, renal insufficiency in particular) (Gomes T. et al. PLoS Med 2017;14:e1002396). Finally, if the epidemiological profile of the two victims is compatible with the data found in the literature concerning pregabalin misuse (young man, history of addiction for case n o 1), these results are in favor of new patterns of use.

Endorsing Complexity Through Diversity: Computational Psychiatry Meets Big Data Analytics

Brain sciences have recently been argued to be the most data-rich speciality in medicine ( 1 Editorial The power of big data must be harnessed for medical progress. Nature. 2016; 539: 467-468 Crossref Scopus (14) Google Scholar ). The abundance of biomedical data is partly explained by neurosciences’ use of magnetic resonance imaging and microarray-type common variant information. For example, genomics and imaging genetics studies commonly investigate >1,000,000 genetic loci (single nucleotide polymorphisms). When individual subjects are characterized by detailed assessments of whole-genome profiles along with brain structure, function, and connectivity measurements, we depend on large participant sample sizes to draw defensible conclusions from our analytical procedures ( 2 Bzdok D. Nichols T.E. Smith S.M. Towards algorithmic analytics for large-scale datasets. Nat Mach Intell. 2019; 1: 296-306 Crossref PubMed Scopus (37) Google Scholar ). Thus, multimodal data initiatives emerged to respond to these calls by aggregating more extensive and phenotypically rich measurements from thousands of subjects. The UK Biobank ( 3 Sudlow C. Gallacher J. Allen N. Beral V. Burton P. Danesh J. et al. UK Biobank: An open access resource for identifying the causes of a wide range of complex diseases of middle and old age. PLOS Med. 2015; 12e1001779 Crossref PubMed Scopus (3238) Google Scholar ) and Adolescent Brain Cognitive Development (ABCD) ( 4 Casey B.J. Cannonier T. Conley M.I. Cohen A.O. Barch D.M. Heitzeg M.M. et al. The Adolescent Brain Cognitive Development (ABCD) study: Imaging acquisition across 21 sites. Dev Cogn Neurosci. 2018; 32: 43-54 Crossref PubMed Scopus (496) Google Scholar ) studies are currently the largest homogeneously acquired populations of old and young age, respectively. The recent advent of population datasets challenges the way we are used to quantitatively analyzing and drawing insights about psychiatric disorders, including autism spectrum disorder or schizophrenia. Because the biology of these disorders is intertwined with various aspects of population stratification, we need to aim for analyses that put forward major sources of population diversity. Specifically, capturing more multifaceted aspects of disease etiopathology bears the potential to improve diagnosis, risk detection, and treatment choice. Therefore, personalized disease-related predictions will be supported by analysis techniques enabling consideration of ethnicity, height, weight/body mass index, gender identity, handedness/language differentiation/hemisphere dominance, personality/risk aversion, race, or hormone metabolism.