Longitudinal Molecular Study Research Articles

Longitudinal single cell atlas identifies complex temporal relationship between type I interferon response and COVID-19 severity

Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

Is Nature Truly Healing Itself? Spontaneous Remissions and Clonal Replacement in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be curable only through the means of allogeneic HSCT. One of the many fascinating and scientifically instructive aspects of the pathogenesis of this disease is the rare possibility of its spontaneous remission with disappearance of PNH clone and abatement of clinical symptoms, which has always captivated the research community. Due to the orphan nature of the condition, no clinical predictors have been identified so far as harbingers of this phenomenon. In a classical scenario, exhaustion of PNH clone may be associated with reappearance of aplastic anemia (AA), in which PNH clone reflects a semi-maladaptive attempt of recovery. Consequently, one could stipulate that the retraction of PNH clone(s) would have to be associated with a compensatory re-expansion of normal hematopoiesis should normal counts be maintained. The recent insights into the AA/PNH pathobiology shed light onto molecular underpinnings of polyclonal vs oligoclonal hematopoiesis and their dynamics. Here, through application of NGS we attempted to better discern the mechanism of PNH spontaneous remission taking advantage of our internal cohort of PNH patients.Among 92 patients with a diagnosis of hemolytic PNH (M:F ratio 0.88, median age 38 years, range 9-84) 41% were primary PNH (pPNH) while 59% were secondary to AA (sPNH). Overall, patients were clinically followed-up for a median time of 68 months (2-339). Median granulocyte clone size was 73% (22-99) with the majority of cases being classified as having a type III dominant red blood cells (RBCs) clone (80%) while 20% type II. Within this cohort, a total of 3 patients underwent spontaneous remission.UPN1 was a 69-year-old male diagnosed with pPNH at the age of 46 after an episode of deep vein thrombosis. He had been managed with prednisone, transfusions and anti-coagulation because of recurrent thrombotic episodes. Once available, he was started on eculizumab and later continued on ravulizumab. His initial flow cytometry study revealed the presence of a type III RBCs clone of 40% and a granulocyte clone of 89%. After 9 years of anti-complement therapy, the patient's clone started a slow decrease and the most recent study revealed a granulocyte clone of 0.02%. Molecular analysis performed at the time of eculizumab start showed a co-dominant mutational configuration by variant allelic frequency (VAF) with PIGA deletion (p.94_95del; VAF 29%) and a BCOR nonsense (p.Y1446X; VAF 27%). No HLA class I/II mutations were found in two longitudinal samples collected 1 year before and after eculizumab start. However, at the last sequencing performed after the complete disappearance of the PNH clone, the patient developed ASXL1 (p.E635Rfs*, VAF 26%) and ZRSR2 (p.E120Gfs*, VAF 42%) mutations along with retraction of the previous PIGA lesion. No decrease in blood counts was noted.UPN2 was a 58-year-old male initially diagnosed with severe AA at the age of 48 and treated with ATG + CsA. At that time, he had a co-existing PNH granulocyte clone of 28%. After 1 year from IST his PNH clone dropped to 1% and since then has been consistently below 1%. Patient has never received anti-complement therapy. At the time of PNH clone retraction, no HLA class I/II or myeloid driver mutations were found and PIGA mutations were not detectable. However, longitudinal molecular studies performed after disappearance of PNH clone revealed the acquisition of ASXL1 p.Q512X mutation at an initial VAF of 23%, which doubled (45%) at last follow-up 5 years later while normal counts persisted.UPN3 was instead a 59-year-old lady diagnosed with pPNH at the age of 30. She had a granulocyte clone as high as 43% with a type II RBCs clone of 17% and a typical PIGA splice site c.981+1G>A mutation (VAF 15%). She was initially treated with transfusions and steroids and her course was complicated by a cerebral venous sinus thrombosis. Patient was eventually given eculizumab and her PNH clone started decreasing until it vanished (last 0.04%) after 8 years. Analysis of samples prior to and after PNH disappearance did not show any HLA class I/II nor myeloid driver gene mutations with absence of PIGA alterations at last sequencing.PNH spontaneous remissions are rare events. In addition to be replaced by polyclonal hematopoiesis, PIGA clones may be swept by CHIP lesions in myeloid genes (e.g. ASXL1) characterized by improved fitness advantage in a process of Darwinian selection. [Display omitted] DisclosuresMaciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

Real-time autOmatically updated data warehOuse in healThcare (ROOT): an innovative and automated data collection system.

BackgroundThe American Society for Clinical Oncology recently launched the minimal common oncology data elements project to facilitate cancer data interoperability. However, clinical data are often unrecorded in an organized way, and converting them into a structured format can be time-consuming. Clinical Data Warehouse (CDW) is a database that consolidates data from different clinical sources. However, the clinical data extracted from this database include not only structured data but also natural language generated during clinical practice. Therefore, applying these data to a clinical study is challenging because they are unstructured, and unformatted to allow essential content to be found. This study determined how best to organize a huge amount of clinical data to evaluate the upper aerodigestive tract cancers’ clinical features and outcomes, including cancer of the head and neck, esophagus, lung, thymus, and mesothelioma.MethodsThe Real-time autOmatically updated data warehOuse in healThcare (ROOT) uses six main regions to describe the journey of cancer patients. This study, developed an algorithm optimized for each disease category using natural language processing of unstructured data and data capture of structured data. Data from patients diagnosed at the Samsung Medical Center from 2008–2020 were used.ResultsComprehensive clinical data for 67,617 patients across six tumor types: 28,954 with non-small-cell lung cancer, 2,540 with small-cell lung cancer, 30,035 with head and neck cancer, 4,950 with esophageal cancer, 966 with thymic cancer, and 172 with mesothelioma were collected. Additionally, the results of a longitudinal molecular study, including epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) tests, and next-generation sequencing (NGS), were included. Scattered information was integrated and automatically built up to match the cohort, allowing users to capture the most updated test results and treatment outcomes.ConclusionsThis landmark study documented the successful construction of a real-time updating system for medical big data, based on the CDW program.

A longitudinal molecular study of the ecology of malaria infections in free-ranging mandrills.

Unravelling the determinants of host variation in susceptibility and exposure to parasite infections, infection dynamics and the consequences of parasitism on host health is of paramount interest to understand the evolution of complex host-parasite interactions. In this study, we evaluated the determinants, temporal changes and physiological correlates of Plasmodium infections in a large natural population of mandrills (Mandrillus sphinx). Over six consecutive years, we obtained detailed parasitological and physiological data from 100 male and female mandrills of all ages. The probability of infection by Plasmodium gonderi and P. mandrilli was elevated (ca. 40%) but most infections were chronical and dynamic, with several cases of parasite switching and clearance. Positive co-infections also occurred between both parasites. Individual age and sex influenced the probability of infections with some differences between parasites: while P. mandrilli appeared to infect its hosts rather randomly, P. gonderi particularly infected middle-aged mandrills. Males were also more susceptible to P. gonderi than females and were more likely to be infected by this parasite at the beginning of an infection by the simian immunodeficiency virus. P. gonderi, and to a lesser extent P. mandrilli, influenced mandrills’ physiology: skin temperatures and neutrophil/lymphocyte ratio were both impacted, generally depending on individual age and sex. These results highlight the ecological complexity of Plasmodium infections in nonhuman primates and the efforts that need to be done to decipher the epidemiology of such parasites.

MNGI-11. LONGITUDINAL GENOMIC ANALYSIS OF SPORADIC MENINGIOMAS WITH MULTIPLE RECURRENCES

Recently, genomic patterns and methylation profiles of meningiomas were described as prognostic factors for aggressiveness and recurrence (e.g. TERT, SMO and AKT1 mutations). However, the particular molecular profiles of recurrence and malignant progression of specific meningioma subtypes remain undefined. In this regard, longitudinal molecular studies are very interesting. In this study, we characterized somatic mutations in a cohort of sporadic meningioma patients. We included 9 patients with multiple recurrences and up to 11 subsequent resections of sporadic meningiomas. We performed a comparative whole-exome sequencing (WES). Moreover, we used the whole-exome data to predict the HLA MHC class I peptides of the subsequent tumor samples in silico. Our study included patients with follow-up times accounted for up to 26 years (average: 12.6 years, range 3–26). We detected a TRAF7 mutation (c.1189G>C) in only 2 of 6 recurrent tumor samples of a female patient with a malignant progression from a WHO I to a WHO grade II meningioma. In a male patient with 9 analyzed meningioma tumor samples, recurrent somatic PLEKHG5, AGBL1, ALK, RICTOR, DEPDC5 mutations were detected. This individual pattern remained constantly evident under different treatments, even after systemic treatment with the mTOR inhibitor everolimus. Moreover, unique mutations in HLA-DQA1, OR14J1, PRH1 genes occurred after mTOR inhibition in this male patient. Furthermore, we identified somatic mutations in the tumor suppressor candidate NBPF1 in 7 of 9 patients. HLA allele types were predicted in silico, with A*02:01, B*57:01, C*03:03, C07:01 and B51:01 being most frequent. Neither ubiquitously shared somatic mutations nor shared MHC class I peptides were identified among this cohort. Taken together, our study outlines a heterogeneous molecular profile in this analysis. Since longitudinal samples in sporadic meningioma are rather rare, the ICOM consortium would be an ideal frame for studying the molecular profile of recurrence in meningioma in larger cohorts.

P06.02 Longitudinal cervicovaginal microbiome measurements of women before and after hiv-seroconversion

Introduction Bacterial vaginosis (BV) by Nugent scoring is associated with enhanced acquisition and cervicovaginal shedding of HIV, but longitudinal molecular studies of these relationships are scarce. Methods HIV-negative (n = 397) female sex workers in Kigali, Rwanda, were followed for two years. Demographic, behavioural, clinical, HIV, sexually transmitted infection, and cervicovaginal microbiota data were collected at regular intervals. The cervicovaginal microbiota were characterised by Nugent scoring, Amsel criteria (pH >4.5, a positive whiff test and presence of >20% clue cells on wet mount; two or three criteria indicated BV diagnosis), and phylogenetic 16S DNA microarray. Results During follow-up, 19 women seroconverted for HIV. The associations between BV by Nugent or Amsel criteria and subsequent HIV seroconversion did not reach statistical significance (aOR = 1.56 (95% CI 0.51–4.77) and aOR = 4.85 (95% CI 0.59–39.90), respectively). For 10/19 women, phylogenetic microbiome composition was available before and after seroconversion, with a median of 324 days (range 42–386) before and 196 days (range 121–492) after seroconversion. Before seroconversion, none of the women had a L. crispatus-dominated microbiome, four had a L. iners -dominated microbiome, four a moderately dysbiotic microbiome and two severe dysbiosis. The microbiome composition of five women remained stable before and after seroconversion, four shifted to (more severe) dysbiosis, and one shifted from dysbiosis to a L. iners -dominated microbiome. After seroconversion, phylogenetic microbiome composition was available for all 19 women. 26% had a L. iners -dominated microbiome, 32% moderate dysbiosis, and 42% severe dysbiosis. Conclusion In this population of high risk women, the association between BV and subsequent HIV seroconversion did not reach statistical significance, but statistical power was limited. The molecular microbiome analysis showed that high levels of L. crispatus may protect against HIV acquisition and that recently acquired HIV infection may make women more prone to dysbiosis. More research is needed to confirm these relationships and determine causality. Disclosure of interest statement This work was funded by the Aids Fonds Netherlands (project number 201102), European and Developing Countries Clinical Trials Partnership (project number CT.2005.33070.001), and the European Commission (CHAARM consortium). The authors report no conflicts of interest.

Enterovirus RNA in longitudinal blood samples and risk of islet autoimmunity in children with a high genetic risk of type 1 diabetes: the MIDIA study.

Only a few longitudinal molecular studies of enterovirus and islet autoimmunity have been reported, and positive results seem to be limited to Finland. We aimed to investigate an association between enterovirus RNA in blood and islet autoimmunity in the MIDIA study from Norway, a country which largely shares environmental and economic features with Finland. We analysed serial blood samples collected at ages 3, 6, and 9 months and then annually from 45 children who developed confirmed positivity for at least two autoantibodies (against insulin, GAD65 and IA-2) and 92 matched controls, all from a cohort of children with a single high-risk HLA-DQ-DR genotype. Enterovirus was tested in RNA extracted from frozen blood cell pellets, using real-time RT-PCR with stringent performance control. Out of 807 blood samples, 72 (8.9%) were positive for enterovirus. There was no association between enterovirus RNA and islet autoimmunity in samples obtained strictly before (7.6% cases, 10.0% controls, OR 0.75 [95% CI 0.36, 1.57]), or strictly after the first detection of islet autoantibodies (10.5% case, 5.8% controls, OR 2.00 [95% CI 0.64, 6.27]). However, there was a tendency towards a higher frequency of enterovirus detection in the first islet autoantibody-positive sample (15.8%) compared with the corresponding time point in matched controls (3.2%, OR 8.7 [95% CI 0.97, 77]). Neither of these results was changed by adjusting for potential confounders, restricting to various time intervals or employing various definitions of enterovirus positivity. Positivity for enterovirus RNA in blood did not predict the later induction of islet autoantibodies, but enterovirus tended to be detected more often at the islet autoantibody seroconversion stage.

Differential findings regarding molecular epidemiology of tuberculosis between two consecutive periods in the context of steady increase of immigration

The demographic characteristics of the population of Madrid, with a steady increase in immigrants, from 4.7% in 1998 to 17.4% in 2007, provide an opportunity to study in depth the transmission of TB. Our aim was to compare two 3-year longitudinal molecular studies of TB to define transmission patterns and predictors of clustering. Two prospective population-based molecular and epidemiological studies (2002–2004 and 2005–2007) of TB patients were conducted in nine urban districts in Madrid using the same methodology. During the period 2002–2007, 2248 cases of TB were reported, and the incidence decreased from 23.5 per 100 000 in 2002 to 20.8 in 2007 (p <0.001). A total of 1269 isolates were molecularly characterized and included in the study. The comparison between the two periods showed that the percentage of foreign-born patients among TB cases increased from 36.2% to 45.7% (p <0.001). Furthermore, the percentage of clustered cases decreased (36.6% vs. 30.6%; p 0.028), and this decline was associated with a decrease of clustered cases among men and people under 35 years. We also observed a decrease in cases belonging to clusters containing >6 people (14.2% vs. 8.2%; p <0.001), and in cases belonging to mixed clusters containing Spanish-born and foreign-born patients (18.5% vs. 11.1%, p <0.001). Our molecular epidemiology study provides clues to interpret the decrease in the incidence of TB in a context of steady increase of immigration. In our region, the decrease in the incidence of TB can be explained predominantly as a result of a decline in recent transmission.

Detectability of Plasmodium falciparum clones

BackgroundIn areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed.MethodsA longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion.ResultsThe distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted.ConclusionsA simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week apart as statistically independent.