Natural History Study Research Articles

Evaluating the global cortical Centiloid value for predicting functional decline

AbstractBackgroundPrevious results on amyloid‐PET imaging for predicting functional decline are conflicting. Given that the Centiloid approach (CL) may serve for inclusion in anti‐amyloid trials, we investigated the extent to which CL may predict functional decline in the AMYPAD‐Prognostic and Natural History Study (PNHS), including 1094 participants.MethodWe analyzed, cross‐sectionally and longitudinally, the associations between CL and global and sum‐of‐boxes (SOB) Clinical Dementia Rating (CDR), and Instrumental Activity of Daily Living (IADL) scores. Mean follow‐up duration ranged from 2.7‐3.0 years (±1.1‐1.6) depending on the functional score. Longitudinal CDR‐SOB and IADL changes were modeled using linear mixed‐effects models with random intercepts and slopes, covarying age and sex, stratified by CDR global score. Analyses included comparisons between three CL groups (Aβ‐: CL<12; grey‐zone: CL = 12‐50; Aβ+: CL>50) and correlation between the baseline CL and the time for progression on the CDR global score.ResultThe baseline CL was significantly higher in CDR = 0 (Mdn = 6.6) than in CDR = 0.5 participants (Mdn = 17.2; p<.001). Baseline CDR‐SOB was significantly higher in Aβ+ (M = 0.48) than in Aβ‐ (M = 0.12) and grey‐zone participants (M = 0.13, p‐values <.001). Baseline IADL was (marginally) significantly lower in Aβ+ (Mdn = 81.3) than in Aβ‐ (Mdn = 82.8, p = .02) and grey‐zone participants (Mdn = 83.5, p = .08, Table 1). Baseline CL significantly predicted subsequent changes in CDR‐SOB and IADL, both in CDR = 0 and CDR = 0.5 participants, but only in the CL>50 groups (βCL12‐50 = 0.03, p = .32,βCL>50 = 0.14, p<.001 for CDR‐SOB in CDR = 0 participants;βCL12‐50 = 0.19, p = .31,βCL>50 = 1.04, p = .01 for CDR‐SOB in CDR = 0.5 participants;βCL12‐50 = ‐0.002, p = .99, βCL>50 = ‐1.00, p = .08 for IADL in CDR = 0 participants; Fig.1). The participants who progressed from global CDR = 0 to CDR = 0.5 (N = 90) had a higher baseline CL (Mdn = 15.16) than stable participants (Mdn = 5.95, p = .002). However, the baseline CL was not associated with a shorter time of clinical progression (rs= ‐.02, p = .8; Fig.2).ConclusionCross‐sectionally, amyloid burden expressed in CL units was associated with functional outcomes. A baseline CL>50 predicted subsequent functional decline in CDR‐SOB and IADL scores in clinically normal and mildly clinically impaired participants. At lower CL values, no functional decline was observed during a three‐year follow‐up. Clinically normal individuals with increasing global CDR scores had higher baseline CL values, but these values were not associated with faster clinical progression.

A Centiloid cut‐off to help predict true amyloid accumulation

AbstractBackgroundLongitudinal amyloid‐PET‐based imaging endpoints are often included in clinical trials, where they provide critical evidence of treatment efficacy. We assessed the longitudinal variability of the Centiloid (CL) scale and its ability to predict early amyloid accumulation.MethodLongitudinal amyloid‐PET with 18F‐flutemetamol and 18F‐florbetaben (N = 711 at follow‐up 1, time interval: 3.0±1.3years; N = 98 at follow‐up 2, 5.1±0.7years) was conducted in predominantly cognitively unimpaired participants of the AMYPAD Prognostic and Natural History Study (PNHS). Quantification of scans was performed using the MR‐based CL pipeline and visually read (VR) by local certified assessors. Longitudinal change in CL was modelled using linear mixed effects models corrected for age, sex, and education. Subjects were classified based on VR status over time (i.e., Stable VR‐, VR‐Converters or Stable VR+) and based on amyloid accumulation over time (i.e., Accumulator or Non‐accumulator; Accumulator if annualised rate of change [ARC]>95th percentile of a subset of 110 subjects for whom no amyloid accumulation was expected, selection criteria in Table‐1). Finally, a baseline CL cut‐off to identify future Accumulators was derived from a receiver operating characteristic (ROC) curve analysis.ResultParticipants had a median age of 65 years, 57% were females, 41% were APOE‐ε4 carriers, and 18% had baseline VR+ scan (Table‐1). The 95th percentile of ARC in the stable 110 subjects was 2.7 CL/year. Baseline CL was higher in Accumulators compared to Non‐accumulators (24.8 versus 8.7 CL, p<.001). Based on the ROC analysis, the optimal baseline CL threshold to predict Accumulators was 12.1 CL (sensitivity = 67% and specificity = 74%, Figure B‐C‐D), in line with the baseline CL threshold that best predicts VR conversion (11.6 CL). Baseline CL values and ARC were also higher in Stable VR+ and VR‐Converters compared to Stable VR‐ (p<.001), but no significant difference in ARC was found between VR‐Converters and Stable VR+ (Table‐1, Figure A). Results were robust across tracers.ConclusionIncreases over 2.7 CL/year can be indicative of reliable amyloid accumulation. A CL cut‐off of ∼12CL can help identify subjects more likely to accumulate amyloid in the short future. Annualised rates of change in amyloid deposition where highly comparable across tracers.

Amyloid‐PET Centiloid quantification predicts cognitive functioning in a pre‐dementia population: findings from AMYPAD‐PNHS

AbstractBackgroundThe prevention of symptomatic Alzheimer’s disease (AD) is a major endeavor currently in the field, with the prevalence of subjects on the AD continuum (i.e. presence of amyloid‐β [Aβ] pathology) being much higher than previously estimated. However, the extent to which Aβ pathology and its interaction with common risk factors in the aging population relates to disease progression is still limited. The AMYPAD Prognostic and Natural History Study (PNHS) aims to evaluate the value of amyloid‐PET for predicting AD‐related disease progression.MethodA total of 1423 non‐demented subjects were included from the AMYPAD‐PNHS pan‐European cohort (17 sites across 10 Parent Cohorts), for who a baseline [18F]flutemetamol or [18F]florbetaben amyloid‐PET scans that passed quality control was available (Table‐1). Amyloid burden was assessed using the Centiloid (CL) method (whole cerebellum reference region). Independent linear mixed‐effect models were used to assess the predictive value of continuous CL, and its interaction with time, across cognitive domains: mini‐mental state examination (MMSE), memory, attention, language fluency, visuospatial, and executive functions. Years from baseline, age, sex, years of education, cognitive dementia rating (CDR), and APOE‐ε4 were used as independent variables. Clinical follow‐up was 3.4 ± 1.8 years (up to ±10 years) on average.ResultAt baseline, 90% of the subjects were classified as cognitively unimpaired (CDR = 0). Based on CL, most subjects were categorized as amyloid‐negative (59%, 0±9CL), followed by grey‐zone (24%, 25±11CL), and amyloid‐positive (17%, 82±21CL). Baseline CL and its interaction with time were predictive of MMSE scores, memory, attention, and language fluency. Baseline CL, but not its interaction with time, was predictive of executive function. Finally, the interaction of baseline CL with time, but not its main effect, was predictive of visuospatial functioning (Figure‐1). Age and sex were significant contributors to nearly all models.ConclusionHigher baseline CL was associated with lower cognitive performance in all explored domains, suggesting that emerging cerebral amyloid accumulation in non‐demented individuals is predictive of overall cognitive function. Also, higher CL values were predictive of decline across cognitive domains, except for executive function. Further analysis is ongoing, including different confounding factors, longitudinal PET outcomes, and other biomarkers of the disease.

A Centiloid cut‐off to help predict true amyloid accumulation

AbstractBackgroundLongitudinal amyloid‐PET‐based imaging endpoints are often included in clinical trials, where they provide critical evidence of treatment efficacy. We assessed the longitudinal variability of the Centiloid (CL) scale and its ability to predict early amyloid accumulation.MethodLongitudinal amyloid‐PET with 18F‐flutemetamol and 18F‐florbetaben (N = 711 at follow‐up 1, time interval: 3.0±1.3years; N = 98 at follow‐up 2, 5.1±0.7years) was conducted in predominantly cognitively unimpaired participants of the AMYPAD Prognostic and Natural History Study (PNHS). Quantification of scans was performed using the MR‐based CL pipeline and visually read (VR) by local certified assessors. Longitudinal change in CL was modelled using linear mixed effects models corrected for age, sex, and education. Subjects were classified based on VR status over time (i.e., Stable VR‐, VR‐Converters or Stable VR+) and based on amyloid accumulation over time (i.e., Accumulator or Non‐accumulator; Accumulator if annualised rate of change [ARC]>95th percentile of a subset of 110 subjects for whom no amyloid accumulation was expected, selection criteria in Table‐1). Finally, a baseline CL cut‐off to identify future Accumulators was derived from a receiver operating characteristic (ROC) curve analysis.ResultParticipants had a median age of 65 years, 57% were females, 41% were APOE‐e4 carriers, and 18% had baseline VR+ scan (Table‐1). The 95th percentile of ARC in the stable 110 subjects was 2.7 CL/year. Baseline CL was higher in Accumulators compared to Non‐accumulators (24.8 versus 8.7 CL, p<.001). Based on the ROC analysis, the optimal baseline CL threshold to predict Accumulators was 12.1 CL (sensitivity = 67% and specificity = 74%, Figure B‐C‐D), in line with the baseline CL threshold that best predicts VR conversion (11.6 CL). Baseline CL values and ARC were also higher in Stable VR+ and VR‐Converters compared to Stable VR‐ (p<.001), but no significant difference in ARC was found between VR‐Converters and Stable VR+ (Table‐1, Figure A). Results were robust across tracers.ConclusionIncreases over 2.7 CL/year can be indicative of reliable amyloid accumulation. A CL cut‐off of ∼12CL can help identify subjects more likely to accumulate amyloid in the short future. Annualised rates of change in amyloid deposition where highly comparable across tracers.

Dentatorubral-pallidoluysian atrophy natural history and biomarkers study

Dentatorubral-pallidoluysian atrophy natural history and biomarkers study

FC35: Depressive symptom transitions in older adults: effects of psychosocial, behavioral, and clinical factors

Objective:Depression evolves dynamically in old age. Studies of natural history of major depression in older adults suggest that 19–34% recover, 27%–32% remain chronically ill, and approximately 40% experience a fluctuating course. Another way of approaching depression from a longitudinal point of view is by adopting a symptom-based approach, that in addition to the evolution of clinically manifested diagnostic entities, also focuses on transitions involving subclinical/subsyndromal states, although few studies have attempted it. We examined psychosocial, behavioral, and clinical determinants of transitions across states that include no depression, subsyndromal-, and clinical depression.Methods:We used data on 3086 adults aged 60+ from the Swedish National Study on Aging and Care in Kungsholmen, followed for 15 years. Markov-state transition models were used to capture transition patterns, as well as their associated determinants. Death and dropout constituted absorbing states. Depression was diagnosed in accordance with DSM-5; SSD was based on having at least 2 symptoms in the absence of DSM diagnosis. Determinants of transition patterns included index of social connections and support (i.e., psychosocial determinants); smoking, alcohol consumption, and physical activity (behavioral determinants); somatic disease burden and history of depression (clinical determinants).Results:At baseline, 10% of the study population exhibited clinically relevant levels of depressive symptoms. Over a 15-year period, a total of 11,489 transitions were observed. Preliminary results indicate that behavioral factors (primarily smoking) were mostly associated with transitions from no depression to clinical depression, as well as from clinical depression to death. Mostly the same pattern was seen for clinical determinants, although higher burden of chronic diseases and previous depression also increased the likelihood of transition from no depression to SSD. Notably, of high baseline values of social connection and support were found to: 1) lower the likelihood of transitioning from no depression to either SSD or clinical depression; 2) lower the likelihood of transitioning from SSD to clinical depression; and 3) increase the likelihood of transitioning from clinical depression to no depression.Conclusion:Clinical and behavioral factors are mostly implicated in lowering the occurrence of depression, whereas psychosocial factors may also be implicated in recovery.

Abstract C097: Understanding rare cancers in Hispanic/Latinx populations: Insights from the MyPART natural history study of rare solid tumors

Abstract PURPOSE: Rare cancers (<15 cases per 100,000 people/year) are poorly understood, particularly in Hispanic/Latinx populations. The My Pediatric and Adult Rare Tumor (MyPART) network conducts a Natural History Study of Rare Solid Tumors (NHSRT) to better understand rare tumor biology and develop effective therapeutic strategies. We analyzed NHSRT Hispanic/Latinx participants. METHODS: Patients of any age with rare solid tumors are eligible. All participants complete medical and family history forms, patient-reported outcomes (PRO) and provide saliva for DNA. Tumors undergo comprehensive molecular analysis and are discussed in a molecular tumor board. A subset of participants is invited to NIH Clinical Center (NIHCC) to undergo a clinical evaluation, genetic counseling, blood collection, and imaging studies, as indicated. Spanish-speaking participants are offered a medical interpreter. Forms are only available in English. RESULTS: Between January 2019 and December 2020, 16 of 200 participants identified as Hispanic/Latinx. Additionally, two international patients, a 15-year-old from Panama with Medullary Thyroid Carcinoma (MTC) who underwent total thyroidectomy with lymphadenectomy, and a 19-year-old from Mexico with Fibrolamellar Carcinoma (FLC) who underwent left hepatectomy and lymphadenectomy were enrolled later and received surgical treatment at NIHCC, which was not accessible in their home countries. Tumor types were neuroendocrine tumors (NET) (n=7), wild-type gastrointestinal stromal tumors (wt-GIST) (n=4), adrenocortical carcinoma (ACC) (n=1), MTC (n=3), and FLC (n=1). Two participants were relatives of patients with an SDHA mutation. Females accounted for 66% of the cohort; the median age was 35 years (range 15-69), enrolled from five US states and five countries. Nine patients used interpreter services. The median time from diagnosis to enrollment was 5.3 years. Half had metastatic disease at diagnosis. At enrollment, 57% of cases with local disease at diagnosis had progressed to metastasis, and 20% showed no evidence of disease. Twelve patients had successful tumor sequencing, and eight were found to have Pathogenic/Likely Pathogenic (Path/LP) variants. MEN1, SMAD4, and LZTR/RPS6KB2 in NET; SDHC in GIST; CDKN2B loss in ACC; DNAJB1-PRACA fusion in FLC; and RET mutations in MTC. Of 12 participants with germline mutation testing, Path/LP variants were found in 10, including SDHA, SDHC, and MUTYH in GIST patients and their relatives; PMS2 and RET in MTC; MEN1 and PALB2 in NET; SLC26A2, OTOF, XPC, and DNAH8 in ACC. CONCLUSION: Enrollment and comprehensive data collection were feasible, granting access to specialty care unavailable to several participants. Hispanic/Latinx representation was low, indicating recruitment challenges and the need for targeted strategies. Remote enrollment facilitated international participation. Making all forms available in Spanish is a priority. Ongoing analysis with over 500 NHRST participants will yield further insights. Citation Format: Juan C. Fierro Pineda, Shadin Ahmed, Karlyne Reilly, Robin Lockridge, Margarita Raygada, Barbara Thomas, John Glod, Abby Sandler, Brigitte Widemann, Mary Frances Wedekind Malone. Understanding rare cancers in Hispanic/Latinx populations: Insights from the MyPART natural history study of rare solid tumors [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C097.

ARIA‐E, ARIA‐H, and CAA‐related inflammation: time of reconsiderations?

AbstractBackgroundEvidence from published clinical trials suggests that high‐dose of monoclonal antibodies (mAbs) are needed to significantly remove parenchymal‐deposited Aβ plaques. However, high‐dosing mAbs resulted in a 25‐35% increased risk of amyloid‐related imaging abnormalities‐edema (ARIA‐E) and ARIA‐hemorrhage (ARIA‐H) side events. Although most ARIA‐E are incidentally recognized as clinically silent during pre‐planned safety monitoring MRI scans and suggested to be managed by dose reduction or suspension, it is noticeable that half of ARIA‐E progressed to symptomatic at continued dosing and to associate with late occurrence of ARIA‐H. Of particular importance, a few of them aggravated to potentially severe brain injury or death due to fatal CAA‐related events. Increased evidence from natural history studies suggests that ARIA‐E is the iatrogenic manifestation of cerebral amyloid angiopathy‐related inflammation (CAA‐ri), a rare autoimmune encephalopathy due to raised anti‐Aß autoAbs levels in the cerebrospinal fluid (CSF).MethodNatural history study of CAA‐ri demonstrated that immunotherapy‐related ARIA‐E share several commonalities with the spontaneous occurrence of autoAbs‐related ARIA‐E, including fluid, MRI, and PET biomarker changes and the potential response to treatment outcomes.ResultThe prospective longitudinal monitoring of patients with CAA‐ri provide evidence for a regional and temporal association of ARIA‐E on MRI with focal peaks of neuroinflammation on microglial‐PET imaging. This association was strong in patients with positive CSF markers of AD (ATN positive) and comorbid CAA pathology on MRI, while it was negligible in patients with CAA disease w/o AD (ATN negative). Raised CSF concentrations of anti‐Aß autoAbs were found in all patients at (sub)acute presentation, and reduced within 5 months from the ARIA‐E index event, in parallel with evidence of radiographic resolution of ARIA‐E. At follow‐up MRI scan, the occurrence of new hemorrhagic markers (aka ARIA‐H in trials) were found only in patients with coexisting CAA and AD comorbid pathology. The MRI scales for ARIA commonly used in clinical trials only partially paralleled the clinical, biological, and MRI findings.ConclusionThis suggests that to mitigate and monitor the risk of ARIA, more sensitive, specific, cost‐effective and easy to interpret diagnostic tools and biomarkers than MRI alone will be needed for precision drug‐dose tailoring.

Genomic landscape of patients with germline RUNX1 variants and familial platelet disorder with myeloid malignancy

AbstractFamilial platelet disorder with associated myeloid malignancies (FPDMM) is caused by germline RUNX1 mutations and characterized by thrombocytopenia and increased risk of hematologic malignancies. We recently launched a longitudinal natural history study for patients with FPDMM. Among 27 families with research genomic data by the end of 2021, 26 different germline RUNX1 variants were detected. Besides missense mutations enriched in Runt homology domain and loss-of-function mutations distributed throughout the gene, splice-region mutations and large deletions were detected in 6 and 7 families, respectively. In 25 of 51 (49%) patients without hematologic malignancy, somatic mutations were detected in at least 1 of the clonal hematopoiesis of indeterminate potential (CHIP) genes or acute myeloid leukemia (AML) driver genes. BCOR was the most frequently mutated gene (in 9 patients), and multiple BCOR mutations were identified in 4 patients. Mutations in 6 other CHIP- or AML-driver genes (TET2, DNMT3A, KRAS, LRP1B, IDH1, and KMT2C) were also found in ≥2 patients without hematologic malignancy. Moreover, 3 unrelated patients (1 with myeloid malignancy) carried somatic mutations in NFE2, which regulates erythroid and megakaryocytic differentiation. Sequential sequencing data from 19 patients demonstrated dynamic changes of somatic mutations over time, and stable clones were more frequently found in older adult patients. In summary, there are diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in patients with FPDMM. Monitoring changes in somatic mutations and clinical manifestations prospectively may reveal mechanisms for malignant progression and inform clinical management. This trial was registered at www.clinicaltrials.gov as #NCT03854318.

A 1-year analysis from a natural history study in Chinese individuals with Duchenne muscular dystrophy

A 1-year analysis from a natural history study in Chinese individuals with Duchenne muscular dystrophy

Bone Marrow, Laboratory, and Clinical Features in Pediatric Patients with RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)

Background: Germline RUNX1 mutation is associated with an autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), most commonly myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but lymphoblastic leukemias are also reported. FPDMM is characterized by thrombocytopenia from an early age associated with abnormal platelet function. Herein we report the bone marrow features, in addition to laboratory and clinical findings, of a large pediatric cohort (N=29) with RUNX1-FPDMM. These data have value in guiding early investigation of neonatal/pediatric thrombocytopenia, and baseline bone marrow interpretation to avoid overdiagnosis of MDS or misdiagnosis of immune-mediated thrombocytopenias. Design: The clinical, laboratory, bone marrow pathology, and molecular findings of 29 pediatric patients (<18 years) with germline RUNX1 mutation followed by the natural history study at the National Institutes of Health (NIH) (ClinicalTrials.gov Identifier: NCT03854318), were analyzed. Results : Among the patients, 13 were female and 16 male, in 21 pedigrees, with a median age at bone marrow biopsy of 6 years (range: 17 months to 17 years). Germline RUNX1 mutations were familial in 26 patients and de novo in 3 patients. Easy bruising and propensity for bleeding were common (87%). The median platelet count was 123 x 10 9/L (range: 38 - 244). Abnormal platelet aggregation assay was present in all 9 patients tested. Abnormal transmission electron microscopy of platelets was found in 14/21 (67%) patients tested; the majority had decreased platelet dense granules, with or without decreased alpha granules. Three patients had normal platelet counts. Hematologic neoplasm was identified in 4 patients: one with AML with JAK3 and KRAS mutations; one with B-lymphoblastic leukemia with EBF1::PDGFRB; one with T-lymphoblastic leukemia with a complex karyotype; and one with MDS with excess blasts and no detectable acquired genetic alterations. 3/4 of patients with malignancy had hypercellular marrows. Of the remaining patients, 19/25 (76%) had hypocellular bone marrows for age. The majority had normal numbers of megakaryocytes. Dysmegakaryopoiesis with features overlapping with “dysplasia” (small hypolobated, small with convoluted nuclei and minimal cytoplasm, separation of nuclear lobes) in greater than 10% of megakaryocytes was present in 92% of cases. At least one patient was previously misdiagnosed with immune thrombocytopenic purpura (ITP). 18 patients had eosinophilia in bone marrow sections, six of whom had peripheral blood eosinophilia. Flow cytometric analysis of the marrow commonly showed T-cells with inverted CD4:CD8 ratios (17/22) and increased eosinophils (16/22). The one patient with MDS had increased (10%) immunophenotypically abnormal myeloblasts expressing CD7 and CD123. 23/25 patients had targeted NGS: one patient had a pathogenic mutation in BCOR and the remaining had no pathogenic mutations in genes associated with hematologic malignancy; 15 patients had VUSs reported. At least four patients had large RUNX1 deletions that were not detected by conventional NGS. 23 patients tested had a normal karyotype except one female patient with monosomy X in 11/20 metaphases. Allergy (drug, food and seasonal) was present in 11 patients, and eczema was common. A spectrum of gastrointestinal (GI) symptoms was seen in 11 patients, including constipation, eosinophilic esophagitis, gastroesophageal reflux, and celiac disease. 2 patients were diagnosed with autism, 1 with coarse facies and KDM6B mutation, 1 with brachydactyly syndrome related to PRMT7 mutation, and 1 with non-syndromic hearing loss related to GJB2 mutation. Conclusion: Germline mutation in RUNX1 should be considered in pediatric patients with thrombocytopenia and/or abnormal platelet function and a hypocellular marrow with or without dysmegakaryopoiesis. Dysmegakaryopoiesis in the setting of RUNX1-FPDMM should not be overinterpreted as pediatric MDS without other supporting criteria such as MDS-defining cytogenetic/molecular abnormalities, multilineage dysplasia, or increased blasts. Patients with large deletions in RUNX1 may be missed on routine NGS testing hence proper germline testing in an experienced laboratory is recommended if suspicion is high. Close monitoring is crucial for early identification of emerging myeloid or lymphoid malignancy.

Identifying Pregnancies at Higher Risk for HPA-1a Alloimmunization and Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT): An International, Prospective, Natural History Study

Background and significance Fetal and neonatal alloimmune thrombocytopenia (FNAIT) results from parental incompatibility of human platelet antigen (HPA) and subsequent maternal sensitization. When this occurs, fetal platelet production is impaired and platelet destruction accelerated. FNAIT is the platelet counterpart of Rhesus disease (RhD); however, FNAIT is rarer, more often occurs in first pregnancies, and alloimmunization may be detected as early as the 2nd trimester. Clinical presentation of FNAIT ranges from no/mild symptoms to severe thrombocytopenia and intracranial hemorrhage, which can take place in the second trimester and cause death or lifelong neurological impairment. Severe FNAIT outcomes impose substantial emotional and financial burden on affected families and healthcare systems. Among pregnant HPA-1a-negative women bearing an HPA-1a-positive fetus at risk of alloimmunization, ~27% carry the HLA-DRB3*01:01 gene variant, increasing sensitization risk ~25 fold and thus leading to much higher FNAIT risk. Screening for FNAIT in pregnancy is not routinely performed, unlike in RhD, resulting in failure to identify at-risk pregnancies prior to birth. To prevent severe FNAIT outcomes, we believe at-risk pregnancies must be identified very early in the second trimester, and prophylactic interventions started immediately. Current management of women with existing alloimmunization due to a prior FNAIT-affected pregnancy involves months of weekly antenatal intravenous immunoglobulin with or without steroid administration. Although fetal platelet counts often improve, this therapy is very burdensome to patients. Most data on FNAIT prevalence and risk are from Caucasian populations; studies in non-Caucasian populations have been too small to be informative. This is the first epidemiological study that seeks to identify HPA-1a-based FNAIT in a racially and ethnically diverse international population of pregnant women. Data to be tracked are: frequency of DRB3*0101 in HPA-1bb women; anti-HPA-1a alloimmunization; pregnancy outcomes; and neonatal thrombocytopenia. These data will provide a contemporary control population preceding future single-arm studies of a novel human anti-HPA-1a monoclonal antibody, RLYB212, in prophylaxis of maternal HPA-1a alloimmunization and FNAIT. The virtual elimination of RhD by screening of all pregnancies and administration of prophylaxis in at-risk cases remains one of the most significant medical advances ever achieved and provides a roadmap for FNAIT. Study design and methods Prospective, non-interventional, natural-history study assessing HPA-1a alloimmunization in pregnant women, conducted across multiple European centers in Norway, Netherlands, UK, and Germany, and in the USA. It will provide insights into anti-HPA-1a-mediated FNAIT risk and frequency among Caucasian and non-Caucasian pregnant women. Up to 30,000 women will be screened at gestational weeks 10-14 to enable early identification and follow-up of those women at higher alloimmunization risk, so that in the future, prophylaxis of the higher-risk women could be initiated. Informed consent will be requested for blood-sample collection at the 10-14 week visit for evaluation of FNAIT risk. Inclusion criteria Pregnant women aged ≥18 years Exclusion criteria Prior history of FNAIT or known HPA-1a alloimmunization Statistical methods Demographic, baseline characteristics and discrete variables will be summarized Primary objective To inform frequency of higher FNAIT risk among pregnant women of diverse racial and ethnic characteristics (all comers). (Figure) Secondary objectives Frequency of HPA-1a maternal alloimmunization at Weeks 10-14 postpartum in pregnant women at higher FNAIT risk and outcomes such as: - Spontaneous abortion: non-deliberate fetal death prior to 19 weeks of gestation - Elective abortion: deliberate termination of pregnancy at any time in gestation - Stillbirth: non-deliberate fetal death after 19 weeks of gestation - Premature birth: live birth prior to 37 completed weeks of gestation - Live births: ≥37 completed weeks of gestation Neonatal and severe neonatal thrombocytopenia: platelet count <150 × 10 9/L and <50 × 10 9/L, respectively, within 72 hours of birth Current status Enrolment for clinical-trial NCT05345561 is ongoing and expected to continue for ~3 years.

Natural History and Clinical Outcomes of Patients with Sickle Cell Disease: The Burden of Avascular Necrosis in a British Cohort

Background: The UK “Natural History Study” is a real-world multi-site cohort study of adults living with sickle cell disorder. Since start of recruitment, data for 241 individuals has been validated: mean age 40.5 (std dev 13.9, range 8-79) with 149 (61.8%) female, and genotypes: HbSS (n = 156; 65%), HbSC (n = 73; 30%), HbS/beta(+) thal (n = 11; 5%) and HbSHPFH (n=1; 0.5%). Avascular Necrosis (AVN) is a debilitating long-term orthopaedic complication of sickle cell disorder and in advanced stages leads to joint destruction and the need for orthopaedic surgery. The present survey looked at the baseline prevalence of AVN in our cohort in relation with clinical parameters. Methods: Patients were recruited after written informed consent by participating sites. Clinical history including the presence of AVN was defined as a diagnosis at the time of recruitment. Treatment history, admission rate, laboratory variables, steady state observations were assessed in univariate t-testing and logistic regression. Results: The prevalence of AVN in the cohort was 21% (50/241). 32 patients had prior orthopaedic surgery for AVN (64%). 38/156 HbSS patients (24%) were affected by AVN, of which 15 HbSS patients (40%) had more than one site of AVN involved. Of 73 HbSC patients, 11 had AVN (15%), 5 of whom had more than one site involved (45%). One patient with HbS/beta(+) had documented AVN. The one patient with HbS/HPFH did not have AVN. The median age of patients with a diagnosis of AVN in the cohort was 45 (range 19-79) and 38 (range 18-79) for patients without AVN, respectively. The median age of first orthopaedic surgery was 37 years (range 20-74). We looked in more depth whether clinical parameters were linked to prevalence of AVN in our cohort, see table 1. (HbS/beta(+) and HbS/HPFH not analysed separately). Discussion: The survey illustrates the burden of AVN in our cohort, with 1 in 5 patients affected. The true prevalence is most likely even higher because cases were only included if recorded in the patients' medical history; this also explains the high percentage (64%) of patients with AVN having had orthopaedic surgery. As can be expected, opiate use at home was significantly higher in patients with AVN (despite previous surgery), in particular among patients with HbSC. AVN was more prevalent in HbSS than other genotypes. There was a trend towards a higher prevalence of AVN in smokers than in non-smokers in the overall group (Odds ratio 2.43, 95%CI 0.90 - 6.53) Within the limitations of the survey, there was a surprising lack of correlation between presence of AVN and disease parameters other than genotype which may be related to sample size. In the HbSS group, there was evidence of a lower haemolytic rate among patients affected by AVN, in keeping with previous reports. The use of hydroxycarbamide at the time of recruitment was not linked to the presence or absence of AVN. Also, HbF levels (regardless of whether patients were taking hydroxycarbamide or not) did not appear to affect the risk of having AVN. Conclusion: Our study prompts further research into prevention of AVN or reversal of early AVN. Traditional risk factors failed to identify those at risk, and longitudinal follow-up as well as biomarkers and genome-wide association studies may help understand the clinical variability of avascular necrosis in sickle cell disorder.

Binary Gender Differences in Quality of Life and Social Supports Among Individuals with Immune Thrombocytopenia (ITP): Data from the ITP Natural History Study Registry

BACKGROUND AND AIMS Immune thrombocytopenia (ITP) is an autoimmune bleeding condition characterized by low platelet count and a range of bleeding risks. Patients with ITP often report their quality-of-life (QoL) is negatively impacted by fatigue, mental health, and changes in their physical appearance. Research on impact of sex differences on QoL for ITP patients is limited. Studies show the importance of social support systems for patients with rare disease, but variation in social support between male and female ITP patients have not been reported. Using patient inputted registry data, we highlight binary gender differences in social support and ITP disease impact on survey participants overall health related QoL. METHODS This is a prospective cohort study of self-reporting adults with ITP registered in the ITP Natural History Study Registry. Data were collected using responses from three surveys within the Registry up to July 3, 2023. Participants were included in the study if they completed all three surveys. Data was analyzed with descriptive statistics and chi-squared tests. Majority of questions asked were based on a Likert scale. Responses were divided into the following categories: ‘Social Supports’, ‘Physical health’, ‘Emotional health’, and overall health related QoL (HRQoL). RESULTS Four hundred and sixty participants were included in the study; 358 (78%) were females over 18 years of age and 102 (22%) were males over 18 years of age. Most participants were from the US (382; 83%) and between ages 18-100 years (mean age 47 years). Social Supports: Women and men reported similar levels of social support.Within the prior seven days, 280 (90%) females and 80 (79%) males reported they had someone to listen to them ‘often’ or ‘always’ (n = 354 (females), 102 males). Two hundred and seventy-nine (78%) females and 77 (72%) males reported they had someone to confide in ‘often’ or ‘always’ (n = 354 (females), 102 males), and 233 (63%) females and 71 (71%) males reported they had someone in their life who understands them ‘often’ or ‘always’ (n = 355 (females), 102 males). Physical health: Women reported significantly more limitations in activities due to physical health compared to men (85; 23% vs 8; 8%; p = .0004) (n = 358 (females); 102 (males). Women reported higher average fatigue levels (109; 19% vs 15; 14%; p = .0015), and greater frequency of feeling tired compared to men (207; 58% vs 27; 27%; p = .00001). Emotional health: Women reported significantly higher levels of negative mental health ‘often’ or ‘always’ impacting mood and ability to think compared to men (201; 57% vs 38; 38%; p = .000618) (n = 356 (females); 102 (males), and reported being bothered ‘often’ or ‘always’ by emotional problems such as feeling anxious, depressed, or irritable, more than men (76; 21% vs 12; 12%; p = .032). Overall HRQoL: Men reported overall HRQoL as either ‘very good’ or ‘excellent’ more than women (179; 50% vs 59; 59%). The difference was not statistically significant. CONCLUSION Our study demonstrates adult females and males with ITP have differences in reported HRQoL. Both reported similar levels of social support, such as having someone who understands their problems and can listen. Despite this, females reported higher frequencies of being limited in doing various activities, feeling tired, mental health struggles, and emotional problems. This study offers novel insight into experiences between adult women and men with ITP. Comparisons with non-ITP participants would be helpful to include in future studies to investigate whether sex differences in HRQoL are in fact due to the ITP itself. Future treatment plans may consider which HRQoL factors are impacted most by ITP and could tailor management approaches accordingly.

Functional Classification of Germline RUNX1 Variants Identified in Patients Suspected of Familial Platelet Disorder with Associated Myeloid Malignancies

RUNX1 is a master regulator of hematopoiesis. A highly conserved runt-homology domain (RHD) located near the N-terminus is responsible for DNA binding and interaction with its heterodimeric partner CBFβ, whereas the C-terminus contains the transactivation domain (TAD). Somatic variants and chromosomal abnormalities affecting RUNX1 are commonly observed in hematological malignancies, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Heterozygous germline RUNX1 variants are responsible for familial platelet disorder with associated myeloid malignancies (FPDMM), an autosomal dominant disorder with incomplete penetrance and a broad spectrum of clinical phenotypes, including thrombocytopenia, functional platelet defects, and an increased risk (35-45%) of developing MDS and AML. In 2019, our group launched a longitudinal natural history study of patients with germline RUNX1 variants to increase our understanding of FPDMM, which will hopefully lead to better clinical management and potentially new therapies. The majority of RUNX1 variants are private with limited segregation data available, making variant interpretation challenging. A significant number of cases have variants of unknown significance (VUS) in RUNX1. Therefore, it is important to determine if these VUS are pathogenic (or benign) in order to improve the diagnosis and clinical management for these individuals. To functionally characterize each variant, we used well-established in vitro functional assays to assess the damaging effect of each RUNX1 VUS, including transactivation reporter assays, electrophoretic mobility shift assays, co-immunoprecipitation, immunofluorescence, and western blot with cell fractionation. All assays include RUNX1 wildtype and known benign and pathogenic controls, as well as co-expression with its binding partner CBFβ. The VUS Ser94Arg located in RHD of RUNX1 has showed impaired CSF1R transactivation (< 20% of wildtype), reduced subcellular co-localization of RUNX1 with CBFβ, and reduced heterodimerization with CBFβ. These results together with population frequency data, in silico prediction, phenotype and segregation data support the reclassification of this variant into the likely pathogenic category. On the other hand, the VUS Asp123His shows no damaging effect in vitro, and could be reclassified to likely benign. Using these assays, we have confirmed or reclassified several RUNX1 variants using the criteria set by the ACGM Myeloid Malignancy VCEP (Luo et al, Blood Adv. 2019; 3:2962). Together, these results highlight the importance of using in vitro functional assays for RUNX1 variant classification. Moreover, we sought to determine the potential of using platelet transcriptomics for RUNX1 variant classification and to gain a deeper understanding of FPDMM pathogenesis. Clariom™ D microarrays identified 218 differentially expressed genes (DEG; absolute fold change ≥ 2; q-value < 0.05) between FPDMM patients and healthy controls, with 90 genes (41%) being upregulated and 128 genes (59%) being downregulated in patients. Hierarchical clustering showed that the selected FPDMM patients with pathogenic and likely pathogenic RUNX1 variants clustered together and separately from healthy controls, with well-established pathogenic variants, such as Arg201Ter and Arg201Gln, at the opposite end of the dendrogram, and likely pathogenic variants such as Tyr403Cysfs153 closer to healthy controls. Our results show that FPDMM patients exhibit reduced expression of important genes related to thrombocytopenia and platelet dysfunction. Furthermore, several top upregulated genes identified in FPDMM patient's platelets are associated with poorer outcomes in AML patients, according to the TCGA dataset. Overall, our study shows the importance of performing functional assays to aid RUNX1 variant classification and to facilitate the study of FPDMM pathogenesis. We are currently analyzing the platelet proteome data of FPDMM patients by LC-MS/MS, which will be reported at the annual meeting.

Persistent Albuminuria and Chronic Kidney Disease in Adults with Sickle Cell Disease: Results from a Multicenter Natural History Study

Background: Patients with sickle cell disease (SCD) are at high risk for developing kidney disease. Albuminuria, an early marker of kidney injury, may be initially transient in patients with SCD. Existing guidelines propose confirming albuminuria at multiple timepoints before intervention. A recent study suggests a urinary albumin-creatinine ratio (UACR) ≥ 100 mg/g correlates highly with persistent albuminuria and subsequent decline in estimated glomerular filtration rate (eGFR). We examined a cohort of patients with SCD to assess those characteristics associated with persistent albuminuria and chronic kidney disease (CKD). Methods During routine clinical visits, we enrolled patients with HbSS or HbSß 0 thalassemia between 18 and 65 years at 4 centers into a prospective study to assess the natural history of kidney disease in SCD. We excluded patients if they had bone marrow transplantation, cancer, connective tissue disease, diabetic nephropathy, other glomerular diseases, hepatitis (B or C), HIV, or were on dialysis. Blood and urine were collected for routine clinical laboratory assessments. Serum cystatin C was measured by commercially available ELISA, and eGFR was calculated using the CKD-EPI-2012 cystatin C equation. We defined CKD as an eGFR < 60 mL/min/1.73m 2 or presence of albuminuria. Glomerular hyperfiltration was defined as an eGFR>130 in females and >140mL/min/1.73m 2 in males, respectively. Persistent albuminuria was considered present if 2 or 3 UACR values were ≥30 mg/g or if initial baseline value was ≥100 mg/g (PMID: 32289161). We utilized generalized linear models adjusting for age and sex to identify characteristics associated with persistent albuminuria and CKD. For multivariable selection, we fit binomial or multinomial LASSO regression models adjusted for age, sex, hemoglobin, %Hemoglobin F, hemoglobinuria, and hydroxyurea to identify those variables associated with outcomes of interest. Results Of 284 enrolled patients, 268 had complete data for UACR, cystatin C and serum creatinine at baseline; 104 (38.8%) of these had persistent albuminuria (Table 1). CKD was present in 40.7% of patients and 26.5% exhibited hyperfiltration. In analyses adjusted for age and sex, several factors were associated with the likelihood of persistent albuminuria. Higher hemoglobin, higher %Hemoglobin F, higher serum bicarbonate and use of NSAIDs were associated with decreased likelihood of persistent albuminuria. Higher systolic and diastolic blood pressure, elevated serum alkaline phosphatase, total bilirubin, and white blood cell count (WBC) were associated with an increased likelihood of persistent albuminuria as were presence of hemoglobinuria and use of angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB). In multivariable selection, higher systolic and diastolic blood pressure, ACEi/ARB use, leg ulcers, higher WBC, higher aspartate transaminase (AST) remained associated with higher likelihood of persistent albuminuria. Presence of avascular necrosis, NSAID use, and higher serum bicarbonate were associated with lower likelihood of persistent albuminuria. When evaluating baseline UACR, individuals with UACR≥100 mg/g had similar probability of subsequent albuminuria as compared to those with severe albuminuria (UACR≥300 mg/g) [Table 2]. Conclusion Persistent albuminuria in this cohort was associated with multiple clinical features. Lower use of NSAIDs in this cohort likely represents counseling of patients to avoid these due to their kidney disease. The relationship with serum bicarbonate suggests a propensity for metabolic acidosis in these patients which could exacerbate the underlying SCD. These data confirm the predictive value of a single UACR value ≥100 mg/g for persistent albuminuria, suggesting this may be a sufficient trigger to warrant intervention. CKD, hyperfiltration and albuminuria were extraordinarily common, consistent with prior studies and validate use of this cohort for additional longitudinal assessment of kidney disease in SCD.

Characterization of the Cynomolgus Macaque Model of Marburg Virus Disease and Assessment of Timing for Therapeutic Treatment Testing.

Marburg virus (MARV) causes severe disease and high mortality in humans. The objective of this study was to characterize disease manifestations and pathogenesis in cynomolgus macaques exposed to MARV. The results of this natural history study may be used to identify features of MARV disease useful in defining the ideal treatment initiation time for subsequent evaluations of investigational therapeutics using this model. Twelve cynomolgus macaques were exposed to a target dose of 1000 plaque-forming units MARV by the intramuscular route, and six control animals were mock-exposed. The primary endpoint of this study was survival to Day 28 post-inoculation (PI). Anesthesia events were minimized with the use of central venous catheters for periodic blood collection, and temperature and activity were continuously monitored by telemetry. All mock-exposed animals remained healthy for the duration of the study. All 12 MARV-exposed animals (100%) became infected, developed illness, and succumbed on Days 8-10 PI. On Day 4 PI, 11 of the 12 MARV-exposed animals had statistically significant temperature elevations over baseline. Clinically observable signs of MARV disease first appeared on Day 5 PI, when 6 of the 12 animals exhibited reduced responsiveness. Ultimately, systemic inflammation, coagulopathy, and direct cytopathic effects of MARV all contributed to multiorgan dysfunction, organ failure, and death or euthanasia of all MARV-exposed animals. Manifestations of MARV disease, including fever, systemic viremia, lymphocytolysis, coagulopathy, and hepatocellular damage, could be used as triggers for initiation of treatment in future therapeutic efficacy studies.

Real-World Use of Recombinant Von Willebrand Factor in People with Clinically Severe Congenital Von Willebrand Disease: Interim Analysis of Athn 9: A Natural History Study for People with Severe VWD

Background: People with von Willebrand disease (VWD) present with a spectrum of bleeding patterns and clinical symptoms, independent of subtype (Rugeri. Haematologica. 2007; Kalot. Haemophilia. 2021). In certain situations, prophylaxis may be appropriate, particularly when there is recurrent and severe bleeding (Connell, Blood Advances, 2021). There are limited real-world data on the use of recombinant von Willebrand factor (rVWF) in the United States. Aim: To characterize the safety and real-world effectiveness of routine prophylaxis with rVWF in people with clinically severe congenital VWD. Study Design and Methods:This is an interim analysis of a phase 4, longitudinal, prospective, observational cohort study (NCT03853486). Participants with severe VWD types 1, 2, and 3 (≤30% of normal) or “clinically severe VWD” (≤40% of normal with severe bleeding and requiring prophylaxis with factor concentrates) were identified from the ATHNdataset. The American Thrombosis & Hemostasis Network (ATHN) is the steward of the ATHNdataset, a Health Insurance Portability and Accountability Act-compliant, de-identified patient health dataset containing data from individuals with bleeding disorders receiving care through ATHN affiliates who opt in to contribute. Treatment regimens were at the discretion of each participant's respective bleeding disorder provider. Participants are followed for 2 years from the time of enrollment. Safety was measured using definitions from the European Haemophilia Safety Surveillance program. Bleeding events (BEs) were measured by individual bleeding components and calculated per the International Society on Thrombosis and Haemostasis Bleed Assessment Tool and/or the Pictorial Bleeding Assessment Chart. Health-related quality of life measures, including an ATHN9 rVWF substudy form, were used to describe real-world effectiveness of rVWF treatment. Descriptive statistics were used to analyze study outcomes. This study was not designed to detect differences in safety or efficacy between individual treatment products. Interim Results:As of June 16, 2023, 23 participants enrolled in the ATHN9 study were receiving rVWF; 14 participants have been prescribed rVWF for at least 1 full year, 13 of whom received continuous prophylaxis. This abstract reports on those 13 participants in the first year of the study. Mean (SD) age was 42.9 (29.2) years; 69.2% (n=9) of participants were female. Most participants had no comorbidities. As expected, most participants had a family history of VWD (84.6%; n=11) and no documented VWF inhibitors (84.6%; n=11); inhibitor status for the remaining 2 participants was unknown. Baseline VWF and factor VIII measurements were reported for most participants ( Table 1). The majority had type 2 VWD (84.6%; n=11), 92.3% (n=12) had identifiable genetic sequence defects in VWF, and 30.8% (n=4) had an abnormal VWF multimer profile. As dictated by the local investigator, rVWF prophylactic regimens ranged from 36.6 IU/kg once weekly to 57.6 IU/kg every 3 days; 7 (53.8%) participants received 40-60 IU/kg at a frequency of 2-3 times per week. Twenty-one BEs occurred in 5 (38.5%) of the participants receiving rVWF prophylaxis during the first year ( Table 2). Most BEs were spontaneous (61.9%; 13/21), occurring in the nose (n=7), gastrointestinal tract (n=5), or ankle (n=1). The remaining BEs were associated with activity with no known trauma (n=4), trauma (n=2), or unknown cause (n=2). One participant reported 3 rVWF treatment-unrelated adverse events (AEs) of humerus shaft closed fracture, metabolic encephalopathy, and acute exacerbation of chronic obstructive airways disease. When surveyed about their experience with rVWF, participants reported very or extremely satisfied as follows: 76.9% (n=10) with infusion time, 84.6% (n=11) with breakthrough bleed control time, 46.2% (n=6) with number of infusions required for breakthrough bleed control, and 69.2% (n=9) with frequency of achieving bleed control without concomitant hemostatic medication. Discussion:In participants with clinically severe congenital VWD, continuous prophylaxis with rVWF was associated with minimal AEs. Most participants receiving rVWF prophylaxis experienced no BEs during the first year of the study. Most participants expressed overall satisfaction with the use of rVWF.

Initial Results in a Phase 1b Trial of PB-04 in Sickle Cell Disease Demonstrate Fetal Hemoglobin Induction, Additive Activity with Hydroxyurea, and Improved Red Blood Cell Sickling Parameters

Extensive evidence has shown fetal hemoglobin (HbF) is a major modulator of sickle cell disease (SCD) phenotype. Clinical benefit with any increment of HbF was shown in the NIH-sponsored Natural History Study and the Multicenter Study of Hydroxyurea (HU), with mean HbF increases of 3.6% above baseline (BL), to improve survival, reduce painful vaso-occlusive crises, and recently stroke prevention and improved cardiac and neurocognitive function with HU are reported. As some patients do not tolerate optimal HU doses due to cytopenias, additional inducers suitable for combined use are desirable. Proportions of F-cells and HbF-quantity/cell have been cited as important. PB-04, a repurposed drug with a well-known safety profile, is an oral fetal globin stimulant identified in high throughput screening, which suppresses 4 repressors of the HMG gene promoter, ( BCL-11A, LSD-1, HDAC-3, KLF-1). PB-04 induces fetal globin in nonhuman primates, transgenic mice, with additive effect when combined with HU in SCD erythroid progenitors, and was shown to reduce infarcts in multiple organs with preserved splenic function in transgenic sickle mice. An ongoing Phase 1b trial in beta thalassemia intermedia (BTI) ( NCT04432623) of 3 doses (1,3, or 5 mg/kg QD) given 3 (TIW) or 5 times/week (Wk) for 12 or 24 Wks in 18 courses in 10 subjects demonstrates a favorable safety profile, dose-proportional PK with 5 >3 mg/kg doses and 5 >3 doses/Wk, Median fold-increases from baseline (BL) in F-cells of 4.4x BL (range 2.3-7x), 2.8x F-reticulocytes (1.6-14.8x), and 6.5x (range 3-11x) increases in mean fluorescence intensity (MFI, representing amount of HbF/RBC). Mean increases in % HbF were 6.5%. Evaluation of PB-04 has now begun in 7 subjects with SCD, 19-51 yrs; 3 were receiving HU with HbF responses (12-22%). Adverse events, F-cells, F-reticulocytes, MFI (amount of HbF/RBC or HbF/reticulocyte) by FACS and ImageStream Analysis and % HbF are assessed Q 4 wks; flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM) and sickling kinetics are assessed q 12 wks. In 4 subjects on 3 mg/kg QD TIW for 24 wks, changes observed from BL values included mean increases of 6% F-cells and 27% F-reticulocytes (2-fold) . In 3 ongoing subjects receiving 5 mg/kg QD TIW for 12 wks mean changes above BL are currently 13% F-cells, 15% F-reticulocytes; HbF/cell or HbF/reticulocyte have increased up to 10-fold. Mean % HbF has increased above HU effects in 2/3 pts on the second dose by 5-12.6% (mean 8.8%), producing 81-95% F-cells, and 35-38% HbF. Decline in FA-WB-VCAM) was observed ± HU and with both doses in 3 subjects. Correlation of HbF quantity/cell with RBC survival and sickling dynamics is being explored. These early results with not-yet-optimized doses of PB-04 suggest additive activity with HU, producing significant HbF and F-cell levels, and support further PK-optimized studies in patients with SCD.

Regional Genetic Heterogeneity Among Patients with Pyruvate Kinase Deficiency

Background: Pyruvate kinase (PK) deficiency is a rare, congenital hemolytic anemia caused by homozygous or compound heterozygous mutations in the PKLR gene, with a prevalence of 3.2-8.5 individuals per million in Western populations. Over 350 pathogenic variants in the PKLR gene have been reported, with the majority being missense substitutions. Although their frequency differs across regions and populations, the geographic distribution of PKLR variants has not yet been fully investigated. Aim: To evaluate the PKLR genotype distribution by region among patients (pts) with PK deficiency in the merged real-world database of the Peak Registry (NCT03481738) and PK Deficiency Natural History Study (NHS; NCT02053480). Methods: The NHS was a global, longitudinal, observational study that enrolled pts with PK deficiency from 2014-2017 (study completed May 2020). The Peak Registry is an ongoing global registry which was initiated in 2018 to build on the NHS with expanded pt recruitment and geographic reach, broader data collection, and longer pt follow-up. Comparable data from these two studies were integrated to form a merged database providing enlarged sample size and prolonged follow-up from pts who participated in both studies. This analysis included pts with available genotype data from the NHS and Peak Registry, including those who were siblings; a large Amish cohort of pts carrying the homozygous p.R479H variant was excluded to avoid reporting bias. Genotype classifications were aligned between the two studies. Data were extracted from the merged database of NHS and Peak (data cutoff date: 13May2022) and summarized descriptively by region. Regions were defined as: Northern (N) Europe (Denmark, Ireland, Netherlands, UK); Central (C) Europe (Czech Republic, Germany, Switzerland); Southern (S) Europe (France, Italy, Spain, Portugal); Asia (S Korea, Thailand, Turkey); N America (Canada, US). Results: 362 pts were included in the analysis (N Europe, N=59; S Europe, N=62; C Europe, N=51; Asia, N=23; N America, N=167), 97 (27%) of whom were siblings. Most pts (72%) had compound heterozygous PKLR variants (Table). Homozygous mutations were most common among pts from Asia (15/23; 65%) and were least common among pts from S Europe (8/62; 13%). Almost half of pts from N Europe (49%) had a compound heterozygous missense/missense (M/M) genotype, while a similar proportion from S Europe (44%) had a compound heterozygous missense/non-missense (M/NM) genotype. A total of 724 PKLR variants were identified, including 197 unique variants, 26 of which were newly identified. Most were missense mutations (76%), followed by nonsense (10%), splice site (7%), and frameshift (5%) mutations. The majority of mutated alleles in the cohort were single nucleotide variants (90%), followed by small deletions (4%), large deletions (4%), and insertions (2%). The amino acid variant with the highest observed frequency was p.Arg510Gln, both overall (158/724; 22%), and in N Europe (31/118; 26%), C Europe (27/102; 26%), and N America (92/334; 28%), but it was not observed in Asia (0/46; 0%). The second most common variant overall, p.Arg486Trp (67/724; 9%), was the most frequent in S Europe (31/124; 25%). When stratified by genotype, p.Arg510Gln was the most common missense mutation in homozygous pts in each region (40-67%) except in Asia. In Asia, p.Thr157Ala, p.Lys541Asn, and p.Arg194Pro were the most common mutations overall (4/46; 9%), and also the most common missense mutations in homozygous pts (4/28; 14%). One hundred and seventy-four unique variants were observed in compound heterozygotes. In S Europe (27/102; 26%) and Asia (2/10; 20%), p.Arg486Trp was the most frequently observed missense mutation in compound heterozygote pts. Overall and in all regions except for Asia (where it was absent), p.Glu241Ter was the most common non-missense mutation in compound heterozygote pts (32/254; 13%); this was p.Gly540Ter (3/6; 50%) in Asia. Conclusions: This analysis, which used the largest real-world data source for pts with PK deficiency, confirms the genetic heterogeneity of the disease, showing that the mutation frequency in PKLR varies substantially based on geographic distribution and that the identification of novel genetic variants is relatively common in PK deficiency.