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Review 1: "Epigenetic Liquid Biopsies Reveal Elevated Vascular Endothelial Cell Turnover and Erythropoiesis in Asymptomatic COVID-19 Patients"

Overall, the reviewers evaluated the preprint positively and recognized its potential significance, giving constructive feedback for the authors to strengthen the manuscript.

Published onSep 12, 2023
Review 1: "Epigenetic Liquid Biopsies Reveal Elevated Vascular Endothelial Cell Turnover and Erythropoiesis in Asymptomatic COVID-19 Patients"
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key-enterThis Pub is a Review of
Epigenetic liquid biopsies reveal elevated vascular endothelial cell turnover and erythropoiesis in asymptomatic COVID-19 patients
Epigenetic liquid biopsies reveal elevated vascular endothelial cell turnover and erythropoiesis in asymptomatic COVID-19 patients

The full spectrum of tissues affected by SARS-CoV-2 infection is crucial for deciphering the heterogenous clinical course of COVID-19. Here, we analyzed DNA methylation and histone modification patterns in circulating chromatin to assess cell type-specific turnover in severe and asymptomatic COVID-19 patients, in relation to clinical outcome. Patients with severe COVID-19 had a massive elevation of circulating cell-free DNA (cfDNA) levels, which originated in lung epithelial cells, cardiomyocytes, vascular endothelial cells and erythroblasts, suggesting increased cell death or turnover in these tissues. The immune response to infection was reflected by elevated B cell and monocyte/macrophage cfDNA levels, and by evidence of an interferon response in cells prior to cfDNA release. Strikingly, monocyte/macrophage cfDNA levels (but not monocyte counts), as well as lung epithelium cfDNA and vascular endothelial cfDNA, predicted clinical deterioration and duration of hospitalization. Asymptomatic patients had elevated levels of immune-derived cfDNA but did not show evidence of pulmonary or cardiac damage. Surprisingly, these patients showed elevated levels of vascular endothelial cell and erythroblast cfDNA, suggesting that sub-clinical vascular and erythrocyte turnover are universal features of COVID-19, independent of disease severity. Epigenetic liquid biopsies provide non-invasive means of monitoring COVID-19 patients, and reveal sub-clinical vascular damage and red blood cell turnover.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.


Review: Researchers conducted an analysis of DNA methylation and histone modification patterns in circulating chromatin to study the tissue dynamics in COVID-19 patients. Severe cases displayed elevated levels of circulating cell-free DNA (cfDNA) originating from various tissues, including lung epithelial cells and vascular endothelial cells, correlating with clinical deterioration and hospitalization duration. Asymptomatic/mildly symptomatic patients also showed increased immune-derived cfDNA levels and evidence of vascular endothelial cell turnover, highlighting the potential of epigenetic liquid biopsies for non-invasive monitoring of COVID-19 patients and revealing sub-clinical tissue damage and red blood cell turnover in the disease.

This is a very well written manuscript and at plasma samples from 120 hospitalized or 68 unaffected individuals this analysis is adequately powered. At the time of the assessment (10 Sep 23, 4 pm GMT), however, the supplementary data except supplementary figures 1 and 2 were not available for me for review. The genetical analyses used seem cutting-edge and appropriate although this is outside of my expertise. 
In hospitalized COVID-19 patients, there is significant and relevant increase in cfDNA over controls (Fig 1A). There is unequivocal increase of cfDNA from all blood lineage cells, liver, endothelial cells and lungs (Fig 1D). Reassuringly, the authors did not find lung cfDNA in healthy controls as seen previously (manuscript refs 16, 17). Regarding the contribution of monocytes/macrophages, the study is inherently biased and the low resolution of the reference atlas prohibits subclassifications of circulating versus resident monocytes, this shortcoming has been acknowledged by the authors. 
Based on these findings the authors designed a specific PCR based assay which they employed on the entire cohort of plasma samples. Again, there is striking and significant increase in cfDNA levels from all tissues analyzed. Moreover, cfDNA correlates with COVID-19 severity index and shows prediction potential in identifying COVID-19 patients at risk of deteriorating. 
Lastly, a novel assay for chromation immunoprecipation of cell-free nucleosomes (cfChIP-seq) has been employed confirming both the power of cfChIP-seq and previous reports on heightened interferon response in severe COVID-19 patients (manuscript refs 35-37).
The author’s claims are largely substantiated by the data available to me although there are a few clarifications needed that I have summarized at the bottom of my review. 

Comments and suggestions:

  1. The heightened presence of cfDNA from multiple cell types may be at least in part iatrogenic. A vast amount of literature shows that corticosteroids, front line treatment in severe COVID-19, change cell numbers or turnover, e.g. in red blood cells and red blood cell progenitors (PMID 7904272, 7904272), lymphocytes (PMID 7904272, 7981603), monocytes (PMID 31507614, 35672449), megakaryocytes (PMID 31507614) … Although the study is not sufficiently powered to subclassify 120 COVID-19 patients, a more granular understanding of treatment modalities will help clarify the issue. Comparison or meta-analysis of previous studies will reveal confounders of cfDNA levels. 

  2. Prognostication of future clinical course is strong from monocyte/macrophage-derived cfDNA but more variably so between myeloid subsets. Moreover, it remains unclear whether there is a difference in demographics, pre-COVID-19 morbidity, treatment regimen of the cohorts which should be addressed by multivariate logistic regression analyses. There are ca 30 deteriorated patients and small sample size could limit stratification.

  3. The changes in total or granular cfDNA concentrations between controls, mild/asymptomatic or hospitalized COVID-19 affected individuals are highly significant. However, it is not clear whether n=19 mild/asymptomatic individuals are proportionally mildly affected or asymptomatic which weakens the conclusion of Fig 5. Additionally, extrapolating the presented data from the manuscript body and Fig 5A there is a at-best moderate effect size (Cohen’s d = 0.69, assuming control mean 5.6 SD 3.18 ng/ml, n=68; mild/asymptomatic 7.8 SD 3.2 ng/ml, n=19). Again, more clinical details are necessary. 

  4. As a pathologist I took it a bit personal to read “In the case of solid organs, this [analysis of cell-free DNA methylation] is equivalent to a standard biopsy, with the advantage that information is summed across the entire organ.” As stated by the authors, stratification of resident or circulating monocytes is not feasible as of today. My suggestion is to adjust as follows: “Analyzing cell-free DNA methylation and histone modifications provides a non-invasive and highly sensitive means to characterize cellular turnover or death within organs, along with the gene expression program occurring in cells prior to their demise and the release of cfDNA. Unlike a standard biopsy, this approach offers the distinct advantage of non-invasiveness and the ability to glean comprehensive information about factors such as methylation and gene expression, offering a broader understanding of tissue dynamics.”

  5. The data availability falls below current publishing standards: scripts are patient agnostic and should be uploaded to github or similar (“availability upon reasonable request” is a no-go in top tier journals). If possible, sequence data should be made available publicly (currently “XXX”) or controlled (dbGaP, European genome-phenome database etc.), the supplementary data is largely unavailable and would have resolved part of my suggestions. 

The manuscript is well-written and adequately powered, analyzing plasma samples from both hospitalized and unaffected individuals. It reveals a significant increase in cell-free DNA (cfDNA) in hospitalized COVID-19 patients compared to controls, originating from various cell types and tissues. The study also highlights the potential of cfDNA levels to predict COVID-19 severity and identifies a novel assay for chromatin immunoprecipitation of cell-free nucleosomes (cfChIP-seq) that confirms previous findings on heightened interferon response in severe COVID-19 patients. Improved data availability and transparency would enhance the study's credibility and facilitate further research in this area. 

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