High-sensitivity analysis of clonal hematopoiesis reveals increased clonal complexity of potential-driver mutations in severe COVID-19 patients.

Whether Clonal Hematopoiesis (CH) represents a risk factor for severity of the COVID-19 disease remains a controversial issue. We report the first high- sensitivity analysis of CH in COVID-19 patients (threshold of detection at 0.5% vs 1 or 2% in previous studies). We analyzed 24 patients admitted to ICU for COVID-19 (COV-ICU) and 19 controls, including healthy subjects and asymptomatic SARS-CoV2-positive individuals. Despite the significantly higher numbers of CH mutations identified (80% mutations with <2% variant allele frequency, VAF), we did not find significant differences between COV-ICU patients and controls in the prevalence of CH or in the numbers, VAF or functional categories of the mutated genes, suggesting that CH is not overrepresented in patients with COVID-19. However, when considering potential drivers CH mutations (CH-PD), COV-ICU patients showed higher clonal complexity, in terms of both mutation numbers and VAF, and enrichment of variants reported in myeloid neoplasms. However, we did not score an impact of increased CH-PD on patient survival or clinical parameters associated with inflammation. These data suggest that COVID-19 influence the clonal composition of the peripheral blood and call for further investigations addressing the potential long-term clinical impact of CH on people experiencing severe COVID-19. We acknowledge that it will indispensable to perform further studies on larger patient cohorts in order to validate and generalize our conclusions. Moreover, we performed CH analysis at a single time point. It will be necessary to consider longitudinal approaches with long periods of follow-up in order to assess if the COVID-19 disease could have an impact on the evolution of CH and long-term consequences in patients that experienced severe COVID-19.

Several lines of evidence suggest a mechanistic link between COVID-19 disease and the pre-existing condition of Clonal Hematopoiesis (CH). Clinically, the presence of somatic mutations at a VAF ≥ 2% (≥4% for X-linked gene mutations in males) in genes associated to myeloid malignancies in the blood or bone marrow of individuals without a diagnosed hematologic disorder and without unexplained cytopenia is currently defined has clonal hematopoiesis of indetermined potential (CHIP) [[1]]. CH and CHIP are highly prevalent in the elderly (10–20% in >70 years old individuals) [[2]–[5]]. Mechanistically, CH results from spontaneous occurrence of aging-associated mutations in normal hematopoietic stem cells, selection of variants conferring growth advantage and progressive expansion of one or more cellular clones. Mutations in genes involved in epigenetic regulation such as DNMT3A, TET2 and ASXL1 account for the majority of CH cases [[3]–[5]].

CH is associated with increased blood cancer risk, with a rate of disease progression that is affected by the size of the clone, numbers and type of mutations [[6]]. Notably, CH has been also linked to increased risk of all-cause mortality and cardiovascular diseases (myocardial infarction, stroke, venous thrombosis, chronic obstructive pulmonary disease), with an associated hazard ratio equal or greater than common cardiovascular risk-factors such as smoking, cholesterol levels and hypertension [[7]]. Emerging evidences suggest that the increased risk of cardiovascular disease is due to a stronger inflammatory response induced by the CH-associated mutations, suggesting that CH may be a general factor in age-related inflammation and disease [[9]–[13]]. Similarly, it has been recently reported an association between CHIP and presence of liver inflammation and fibrosis, that double the risk of CHIP carriers of developing chronic liver disease [[14]]. Studies in model systems suggest that mutant macrophages induce an inflammatory milieu which contributes to clonal expansion and associated comorbidities [[14]].

COVID-19 prevalence and/or severity are associated with features that are similar to those observed in CH, including increased severity with age, with a lower percentage of survivors in the 63–76 years old range, and increased incidence of coagulation disorders (disseminated intravascular coagulation and venous thromboembolism) and cardiovascular diseases (cardiac ischemia). Notably, cardiac injury is common in COVID-19 hospitalized patients and COVID-19 is associated with abnormal activation of tissue macrophages and systemic hyper-inflammation [[16]–[21]].

The impact of CH on the incidence and the severity of COVID-19 is still a debated issue, considering that different studies reported opposite results. The data from Bolton et al. [[22]], Duployez et al. [[23]], Li et al. [[24]] and Kang et al. [[25]] showed a higher prevalence of CH in COVID-19 patients and an increased risk for severe COVID-19 disease in presence of CH. In contrast, data from Hameister et al. [[26]], Petzer et al. [[27]] and Zhou et al. [[28]] showed that CH is not overrepresented in COVID-19 patients and its presence does not appear to be relevant for the severity of the disease.

The aim of our study was to analyze the presence and the impact of CH in COVID-19 patients using high sensitivity approaches enabling the identification of low-frequency clones.

Our cohort of patients includes hospitalized patients with confirmed SARS-CoV-2 infection by molecular testing and admitted to intensive care unit (ICU) of the San Gerardo Hospital, Monza Italy, because of severe pneumonia requiring invasive mechanical ventilation. Samples were collected on April 2020 and obtained from an indwelling arterial line. Patients' data were collected as part of the STORM study (Spallanzani Institute approval number 84/2020; NCT04424992). Deferred written informed consent was obtained from all participants.

As controls, we included personnel working at the European Institute of Oncology (IEO), that during the SARS-CoV-2 pandemic was involved in the prospective study SOS-COV2 (IEO 1271) (details and results of the study are published in [[29]]). In particular, 12 individuals scored positive to SARS-CoV-2 infection by qPCR (Swab+, Table 1) and/or by detection of circulating IgGs (IgG+, Table 1). All infected individuals reported pauci- or no-symptoms correlated to SARS-CoV-2 infection and we defined them as asymptomatic positive (asymPOS). 7 individuals tested negative to SARS-CoV-2 infection and were healthy (NEG, Table 1). Ethical approval was granted by the IEO ethical committee (IEO 1271) and written informed consent was obtained from the participants. Peripheral blood samples were collected during the first wave of the SARS-CoV-2 pandemic in Italy from June to September 2020. As controls, we took advantage of the SOS-COV2 study at our Institute in order to avoid as many confounding variables as possible, including demographic similarities and/or exposure risks. Indeed, samples from both cohorts were collected during the same phase of the SARS-CoV-2 pandemic in the same regional area (Lombardy, Italy). This minimizes also the probability of infections from different SARS-CoV-2 viral variants, that clearly showed different microbiological characteristics and, more importantly, completely different clinical impacts on the infected individuals. Moreover, we wanted to analyze controls collected before the vaccination campaign, in order to avoid possible confounding effects imposed by vaccination on the hematopoietic cell system of our individuals.

Graph

Table 1 Main clinical features of the subjects included in our study.

1 NA = Not applicable

2 aApplicable only to ICU patients. Controls are paucisymptomatic, asymptomatic or healthy individuals.

Main clinical features and demographic information of the individuals involved in our study are reported in Table 1 and S1 Table.

CH mutations were analyzed on peripheral blood mononuclear cells by error-suppressed sequencing using a custom gene panel, the CHIP-UMI Panel, which allows analyses of all protein-coding exons of the 80 genes most frequently mutated in 19 clonal hematopoiesis studies (see Supplementary Materials and methods and S1 Table in S1 File). Sequencing libraries were prepared with the SureSelectXT HS Target Enrichment System according to the manufacturer's instructions (Agilent Technologies). Pooled libraries were sequenced on an Illumina Novaseq 6000 with 2x100 bp paired-end reads, obtaining an average coverage of 1350X.

Analysis of the sequencing reads was performed using the Alissa software from Agilent Technologies and aligning to the GRCh38 reference human genome. We filtered for variants affecting the coding sequence: Indels and non-synonymous, stop-gain and stop/start loss SNVs. We filtered for germline polymorphisms, removing any variant reported in any population database with a frequency >0.005 by Alissa Interpret. We next applied a serial of post-processing filters in order to remove further putative germline polymorphisms and potentially false-positive variants introduced by sequencing artifacts. In particular, we used as reference a cohort of 130 samples obtained from individuals enrolled in different studies designed in IEO for the analysis of CH (see Supplementary Materials and methods in S1 File for details). We, finally, validated all variants passing these sequential filters by manual inspection on Integrative Genomics Viewer. The detailed list of mutations identified is reported in S2 Table.

We classified as potential drivers (PD) all CH mutations identified in our study according to the criteria described in [[30]–[32]]. In particular, for classification of somatic variants as drivers we used the criteria described in [[31]]. Moreover, we classified genes as oncogene or tumor suppressor genes according to OncoKB, Cancer Gene Census and other scientific literature and, then, we considered as driver: i) any truncating mutation (nonsense, essential splice site or frameshift indel) in known tumor suppressor genes; ii) any somatic mutation identified in >10 cases in the "haematopoietic and lymphoid" category in COSMIC or in the 8 studies on Myeloid neoplasms of cBioportal (see Supplementary Materials and methods in S1 File for details); iii) any somatic mutation identified in >20 cases in any other category in COSMIC or in all PanCancer Studies of cBioportal.

Statistical analysis for comparison between disease groups was performed using GraphPad Prism software and the non-parametric two-sided Mann–Whitney U test or the one-tailed Z-test. The Mantel–Cox log-rank test was used to compare survival rates. p values <0.05 were considered significant.

To analyze the presence of CH in patients with COVD-19 disease, we performed a high-sensitivity, error-suppressed analyses of CH mutations in 24 hospitalized COVID-19 patients (COV-ICU) and 19 controls, including 12 SARS-CoV-2-positive individuals with asymptomatic infection (asymPOS) and 7 -negative (NEG). Demographic and clinical features of the enrolled individuals are reported in Table 1 and S1 Table. The asymPOS group included the youngest individuals, with the oldest ones all in the COV-ICU cohort. Age of the negative individuals (NEG), instead, was comparable to that of the COV-ICU patients (S1 Fig in S1 File). Despite significant differences in median age, our analysis of CH mutations showed only a modest difference in numbers of mutations identified in the peripheral blood of NEG versus asymPOS individuals, and no significant differences were observed concerning the VAF and genes targeted by CH-mutations (S2 Fig in S1 File). Thus, negative and asymptomatic individuals (n = 19) were considered as a single control group and analyzed together with respect to the group of the COV-ICU patients (n = 24).

The prevalence of CH was 83.3% (20/24) and 74% (14/19) in the COV-ICU and control groups, with only 4 and 5 individuals harboring no CH mutations, respectively. Collectively, we identified 117 CH mutations in 47 genes: 71 in the COV-ICU patients and 46 in the controls, with a median VAF of 1.1% (min 0.5, max 29.5%) and 0.95% (min 0.6, max 5.9%), respectively (Table 2). The vast majority of mutations, 77.5% for COV-ICU patients and 89% for controls, had a VAF <2% (Table 2). As for the CH prevalence, neither number or VAF of CH mutations showed significant differences between COV-ICU and controls (Fig 1A and 1B, Table 2). Likewise, number of individuals harboring >1 CH mutation was similar between the two groups. Indeed, ≥ 3 or more mutations were identified in 12/20 (60%) COV-ICU patients and 9/14 (64%) in controls, with maximum of 9 mutations in one patient in the COV-ICU group and 6 in one control individual (Fig 1C and Table 2). In agreement with the association of CH prevalence and expansion of CH clones with age [[3]–[5]], we observed an increase in the number of CH mutations per patient and their VAF with age. However, these trends were again not significantly different between the two groups (Fig 2). Finally, the mutational landscapes of the two groups were also very similar (S3 Fig in S1 File). Restricting our analysis exclusively to mutations with VAF ≥ 2%, we scored a CH prevalence of 42% (10/24) and 21% (4/19) in COV-ICU patients and controls, respectively, in accordance with previous studies [[22], [26], [28]]. However, this difference was not statistically significant, as for the number (17 vs 5) and the median VAF (2.8% vs 2.6%) of CH mutations in the two groups, confirming the results obtained from the analysis of our complete dataset of CH variants.

Graph: A) Number of CH mutations identified in each individual. B) Variant allele frequency (VAF) of the CH mutations identified. C) Number of patients with the indicated number of CH mutations in our cohort. The horizontal line indicates the mean and the whiskers the SEM. Statistical significance was calculated using Mann–Whitney U test. COV-ICU, patients with severe COVID-19; Controls, control group; ns, not-significant.

Graph: A) Number of CH mutations per individual according to their age. B) Variant allele frequency (VAF) of CH mutations in individuals according to their age. COV-ICU, patients with severe COVID-19; Controls, control group. Boxes define the 25th and the 75th percentiles; horizontal line within the boxes indicates the median and whiskers define the Min to Max002E.

Graph

Table 2 CH mutations identified in our cohort.

To assess their pathogenetic potential, we first annotated the identified CH mutations as putative drivers (CH-PD), according to previously described criteria [[30]–[32]] and as detailed in Materials and methods. Strikingly, we scored higher numbers and significantly higher percentages of CH-PD mutations in COV-ICU patients (22/71 mutations, 31%), as compared to controls (8/46 mutations; 17%) (Fig 3A; p = 0.044). To investigate if the identified CH mutations could potentially increase the risk of developing subsequent pathologies (hazardous mutations), we searched for their frequency in two datasets of mutations containing, respectively, all CH mutations published insofar, and the mutations reported in myeloid neoplasms in cBioPortal (https://www.cbioportal.org/, see Supplementary Materials and methods in S1 File for details). Notably, the COV-ICU cohort contained more hazardous mutations (n = 16; 21%), as compared to controls (n = 3; 6.5%) (p = 0.016; see Table 2 for details).

Graph: A) Number of CH-PD mutations identified in each individual. B) Variant allele frequency (VAF) of the CH-PD mutations identified. C) Number of patients with the indicated number of CH-PD mutations in our cohort. D) VAF of CH-PD mutations in individuals according to their age. The horizontal line indicates the mean and the whiskers the SEM. Statistical significance was calculated using Mann–Whitney U test. COV-ICU, patients with severe COVID-19; Controls, control group; CH-PD, potential driver mutation in CH.

Finally, we analyzed clonal complexity of the CH-PD mutations in the COV-ICU and control groups. None of the control individuals harboring CH-PD mutations showed >1 mutation (0/8; Table 2). Strikingly, 8 of the 12 COV-ICU patients with CH-PD mutations (67%) showed instead >1 mutation, with two patients harboring 3 of them (Fig 3C and Table 2). Most CH-PD had a low VAF, with a median of 1.35% (min 0.7%, max 29.5%; Fig 3B and Table 2). Individual CH-PD mutations in COV-ICU patients showed a higher, albeit statistically not significant, VAF than those in controls (Fig 3B). Importantly, the higher clonal complexity, higher numbers and VAF of CH-PD mutations in the COV-ICU group vs. controls appeared to be age-independent. In the age group 50–59 years old, we observed 2/2 (100%) patients with a total of 3 driver mutations with a mean VAF of 1.8% (range 0.7–2.4%) in the COV-ICU group, as compared to 3/5 (60%) individuals with 3 driver mutations with mean VAF of 0.8% (range 0.6–1.3%) in the control group. As well, in the age group 60–69 years old, we observed 6/11 (55%) patients with 12 driver mutations and mean VAF of 2.8% (range 0.7–10.4%) in the COV-ICU and 4/6 (67%) with 4 driver mutations and average VAF of 1.4% (range 0.9–2.6%) in the controls (Fig 3C and Table 2). Nonetheless, the presence of driver mutations in the COV-ICU patients did not seem to significantly affect their survival or their clinical parameters (S1 Table and S4 Fig in S1 File).

To our knowledge, this is the first study that analyzes the impact of CH on COVID-19 disease with a limit of sensitivity equal to 1% of nucleated blood cells, which is considerably lower to the 4% currently used to define CHIP operatively. Consistently, as compared to the published studies [[22]–[24], [26]–[28]], we identified significantly higher numbers of CH mutations per patient (overall, 2.8 vs <1 mutations) and higher prevalence of CH (overall, >80% vs <50%) and found CH clones also in individuals with <40 years. These data are in agreement with previous studies of CH at high sensitivity in non-COVID individuals, which also reported a higher prevalence of CH in the population, as compared to standard NGS approaches, especially at younger ages [[30], [33]–[36]].

Nonetheless, our data showed similar prevalence of CH, size of CH clones and mutational landscape in COVID-19 patients and controls, ruling out a positive association between CH and incidence and/or aggravation of the infection from SARS-CoV-2, in line with results reported in Hameister et al. [[26]], Petzer et al. [[27]] and Zhou et al. [[28]]. A recent study showed an association between presence of myeloid CH clones and increased risk of mortality due to COVID-19. This association, however, was significant only in the age group of 75–84 years old individuals [[37]]. In our cohort, there are no COVID-19 patients ≥75 years old. Therefore, we cannot exclude that the absence of association between CH and outcome of the COVID-19 disease in our study is due to the younger age of our patients. Nonetheless, interestingly, Del Pozo-Valero et al. reported that CH variants classified as pathogenic or likely pathogenic were significantly more represented in the COVID-19 patients with increased risk of mortality [[37]]. This is in line with our observation, that COVID-19 patients with severe disease harbor more CH-PD and hazardous mutations, as discussed below.

More than 80% of the mutations we identified had VAF<2%. The biological meaning and clinical relevance of these small clones remain to be elucidated. They could simply represent a transient modification of the genomic landscape of the peripheral blood of healthy individuals, highly dynamic and constantly changing. Mutated clones likely change in time quite frequently and, without a specific environmental pressure, most of these mutated clones at low frequency could simply be envisioned as passengers with no particular consequences for the fitness of the cells harboring them, as suggested by recent reports [[38]–[40]]. Regardless of mechanisms and duration of the fitness advantage conferred by low-VAF mutations, their presence might impact different physiological functions, including immunity, thus influencing the penetrance of different disease phenotypes.

It has been reported that CH clones with different mutations expand with different growth rates, which are dependent both on the mutated gene and the specific aminoacidic change: i.e. mutations in DNMT3A show a slower clonal expansion (5% per year) compared to mutations in TET2 (10% per yr) or splicing factors (15–20% per yr). Moreover, mutation fitness appears not to be constant over the life time of an individual: some clones grow more rapidly early in life and then their growth rate decreases during old age (clones with mutations in DNMT3A, BRCC3, TP53), while other clones show no deceleration (clones with mutations in U2AF1, SRSF2P95H, IDH1) and TET2 mutations show a quite constant growth rate and overtake clonal hematopoiesis later in life [[40]]. Considering that the majority of the clones we identified have a VAF below the clinical threshold of 2% and we found the majority of CH-PD mutations in DNMT3A (no in the hotspot R882 position, which is the DNMT3A mutated position that confers the highest fitness to clonal growth), TET2 and ASXL1, we expect that clonal expansion in our patients would require a time frame of several years. The design and development of specific trials with long-term longitudinal follow-up would be instrumental in order to score clonal expansion and define if the CH-PD mutations we identified are bona fide markers of disease progression.

In our cohort, we found 15 CH-PD mutations (14 in COV-ICU and 1 in controls) in common with mutations identified in myeloid neoplasms in the cBioPortal database. Interestingly, some of these mutations had low VAF in patients with myeloid neoplasms, similar to the VAF scored in our study. Namely, DNMT3A p.R899C and BRCC3 p.Q299* have been reported with a VAF = 2% in patients with MDS and were found with a VAF equal to 1.8% and 1% in our COV-ICU patients, respectively. The hotspot mutations GNB1 p.K57E has been reported in several patients with MDS with VAF = 2%. PPM1D truncating variant p.S468*, found with a VAF = 9.7% in ICU16, has been reported with a VAF = 3 and 4% in two patients with MDS. TET2 p.I750Rfs*62 have been reported in a patient with CMML. Furthermore, in a recent longitudinal study, it has been shown that fast growing clones with potentially harmful variants were originally identified has clones with a VAF<2% [[39]]. Altogether these data suggest that clinically relevant variants can be found at VAF that do not reach the clinical threshold of CHIP and that we need to favor more inclusive analysis of CH because significant information can be revealed by low frequency mutations.

Mutations in different CH-genes have been associated to distinct risks of development of subsequent pathologies, in particular acute myeloid leukemia (AML) and coronary heart disease (CHD). For example, mutations in TP53 or in splicing factors (U2AF1, SF3B1, SRSF2) are linked to a particularly high risk of developing AML. Considering CHD, instead, mutations in DNMT3A, TET2 and ASXL1 double the relative risk, while mutations in JAK2 increase the relative risk up to 12 folds (reviewed in [[6]]). Thus, it appears that the type of CH mutations impact on the evolution of potential downstream diseases. In our cohort, in patients with severe COVID-19 disease we observed a significantly higher percentage of CH-PD mutations, most of which (16/22, 73%) in common with datasets of hazardous mutations. Moreover, in COV-ICU patients the clonal complexity for these mutations was higher than in controls, suggesting that their presence might have a clinical impact. We cannot formally exclude the possibility that these CH-PD mutations could confer an increased risk of faster degenerating COVID-19 disease or prolonged illness, may be affecting the host immune responses and their antiviral defenses. However, most notably, in the COV-ICU patients we did not score an impact of the presence of these potential driver mutations on patient survival or clinical parameters associated with inflammation.

Even if the numbers of our cohort are too small to draw definitive conclusions, the lack of an impact of high-frequency CH-PD on clinical-biological parameters argues against an association between CH and COVID-19 prognosis, suggesting that the COVID-19 disease might instead select for hazardous CH-PD mutations. Systemic factors associated to severe disease, such as increased production of inflammatory cytokines, could favor the positive selection of clones with a selective advantage. Under this scenario, clonal expansion could be reversible and the CH clones disappear after clinical recovery from COVID-19, or, alternatively, once a mutation conferring proliferative advantage is acquired, the clones may continue to expand independently from the resolution of the COVID-19 disease, thus increasing the risk of hematological disease or other aging-associated morbidities. To our knowledge, up to date, only three studies analyzed the dynamic of CH clones in paired samples from the same individual at different follow-up: in 9 COVID-19 patients from 7 to 16 days apart, in 8 patients during a 6 months follow-up, and in 54 critically ill patients tested 8 days apart [[23], [27]]. None of the studies reported major changes in VAF of the detected CH variants at the different time points analyzed, suggesting no mayor effects on clonal expansion [[23], [27]].

In conclusion, our data seem to suggest that the COVID-19 disease could influence the clonal composition of the peripheral blood of COVID-19 patients with severe disease. Nonetheless, our study uses a relatively small sample size. It will indispensable to perform appropriate further studies on larger patient cohorts in order to be able to validate and generalize our conclusions. Moreover, we analyzed the PB clonal composition at a single time point. It will be necessary to consider longitudinal approaches to track VAF variations over time with long periods of follow-up in order to assess if the COVID-19 disease could have a long-term impact on the evolution of CH in people that experienced severe COVID-19 and long-term consequences on patient outcomes.

S1 File

(DOCX)

S1 Table

Clinical parameters of COVID19 patients admitted to ICU.

(XLSX)

S2 Table

Detailed list of CH-mutations identified in our study.

(XLSX)

Solimando Antonio Academic Editor

21 Jun 2023

PONE-D-23-04485High-sensitivity Analysis of Clonal Hematopoiesis Reveals Increased Clonal Complexity of Potential-Driver Mutations in Severe COVID-19 PatientsPLOS ONE

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Reviewer #1: Doctors Chiara Ronchini, Pier Giuseppe Pelicci, and colleagues present a study of the prevalence of clonal hematopoiesis (CH)(down to a reported variant allele detection threshold of 0.5%) in 24 patients admitted to the ICU for COVID-19 (COV-ICU), and in 19 controls that include healthy subjects and those with asymptomatic, documented SARS-CoV2 infection. Major findings included the high prevalence of CH in both groups, mainly with VAF <2%, as expected by the error-corrected sequencing approach and the few previous studies that have found CH to be common in adults at VAF less than the CHIP threshold of 2%. When considering CH variants more strongly associated with driving clonal expansion and/or myeloid malignancy (CH-PD or "hazardous mutations"), the authors found a significantly higher prevalence in COV-ICU patients, as compared to controls, along with increased features of clonal complexity. Finally, the authors found no significant impact of the presence of CH-PD on clinical outcomes of ICU patients, including the probability of survival, or blood counts, clinical indicators of hemostasis or oxygen measures.

There have been several publications relating to the prevalence and clinical impact of CH in COVID-19 and the authors acknowledge these. The novelty lies mostly in the application of error-suppressed sequencing and the analysis of CH clones at lower mean VAF than previous published studies.

The major limitation, which the authors also acknowledge, is that the number of participants in their cohort is small and that this limits the ability to draw definitive conclusions. Nevertheless, their observations call for larger studies to examine (or re-examine) the potential impact of more ubiquitous, lower-VAF level CH, and prospective analysis of longer-term outcomes of COVID patients who survive ICU admission, with and without detectable CH.

These additional comments are meant to improve the quality of the manuscript:

Reviewer #2: The authors re-evaluate the link between clonal hematopoiesis and COVID-19 severity by increasing the detection sensitivity of the defining somatic mutations (threshold 0.5%) in 24 patients with severe disease, 12 asymptomatic patients and 9 non-infected donors. The tested gene panel includes 80 genes, the coverage is 1350X, COSMIC and cBioportal are used to identify driver genes with a VAF that, in more than 80% of cases, was in the 0.5-2% range. The total number of detected mutations was similar in both group. Then, potential driver mutations and clonal complexity were analyzed, and these two parameters were higher in severe patients, without significant impact on clinical and biological parameters and disease outcome in this small cohort. Altogether, this study further argue for a limited impact of clonal hematopoiesis on severe COVID-19 outcome. The manuscript is clearly written and complements a series of previous analyses with various conclusions in small cohorts. A meta analysis will be needed for solid conclusions regarding the outcome. This report is a useful contribution to prepare this analysis.

In the introduction, the authors may stick to WHO 2022 classification of CHIP (Khoury et al, Leukemia 2022): CHIP refers to somatic mutations of myeloid malignancy-associated genes detected in the blood or bone marrow at a VAF ≥ 2% (≥4% for X-linked gene mutations in males) in individuals without a diagnosed hematologic disorder and without unexplained cytopenia.

The WHO classification also defines clonal cytopenia of undetermined significance (CCUS) as CHIP in the presence of one or more persistent cytopenias that are otherwise unexplained by hematologic or non-hematologic conditions and that do not meet diagnostic criteria for defined myeloid neoplasms. Did some of the patients included in this trial demonstrate a cytopenia before inclusion?

The introduction also focuses on the cardiovascular risk of CHIP but many other risks have now been identified, the latest being liver diseases (see B Ebert's paper in Nature recently)

Since they were included in 2020, do the authors have some information regarding the long-term outcome of the survivors?

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4 Aug 2023

We thank the Reviewers for their valued comments and for highlighting some issues, we acknowledge that they helped us improving the quality of our manuscript.

In the following pages are our point-by-point responses (line # according to the revised manuscript without track changes).

Reviewer #1: Doctors Chiara Ronchini, Pier Giuseppe Pelicci, and colleagues present a study of the prevalence of clonal hematopoiesis (CH)(down to a reported variant allele detection threshold of 0.5%) in 24 patients admitted to the ICU for COVID-19 (COV-ICU), and in 19 controls that include healthy subjects and those with asymptomatic, documented SARS-CoV2 infection. Major findings included the high prevalence of CH in both groups, mainly with VAF <2%, as expected by the error-corrected sequencing approach and the few previous studies that have found CH to be common in adults at VAF less than the CHIP threshold of 2%. When considering CH variants more strongly associated with driving clonal expansion and/or myeloid malignancy (CH-PD or "hazardous mutations"), the authors found a significantly higher prevalence in COV-ICU patients, as compared to controls, along with increased features of clonal complexity. Finally, the authors found no significant impact of the presence of CH-PD on clinical outcomes of ICU patients, including the probability of survival, or blood counts, clinical indicators of hemostasis or oxygen measures.

There have been several publications relating to the prevalence and clinical impact of CH in COVID-19 and the authors acknowledge these. The novelty lies mostly in the application of error-suppressed sequencing and the analysis of CH clones at lower mean VAF than previous published studies.

The major limitation, which the authors also acknowledge, is that the number of participants in their cohort is small and that this limits the ability to draw definitive conclusions. Nevertheless, their observations call for larger studies to examine (or re-examine) the potential impact of more ubiquitous, lower-VAF level CH, and prospective analysis of longer-term outcomes of COVID patients who survive ICU admission, with and without detectable CH.

These additional comments are meant to improve the quality of the manuscript:

Response: The sequencing approach we used for this study with the CHIP-UMI panel has not been published yet. The manuscript about the validation of our approach is in preparation. We added some details on the performance of the method in the Supplementary Materials and Methods section and we changed the nomenclature to error-suppression, as suggested. We are well aware of the issue of sequencing artifacts and that is why we created a database of "Likely-FalsePositive", which we are constantly updating with new sequencing data from new individuals with the CHIP-UMI panel. Since the submission of the paper to PLOSone we increased the number of sequenced samples with the CHIP-UMI from 91 to 130.

We carefully reevaluated all CH variants identified in our study and, in particular, the ones highlighted by the Reviewer. We removed from our dataset ASXL2 c.3900C>G and ATRX c.5424 T>G, because with the new analysis they were found in ≥3 individuals and are, therefore, potential artifacts. We revisited all our data and analysis and updated all tables and figures, accordingly.

Concerning the ASXL1 c.1934dupG (p.G646Wfs*12) variant, we are aware of the conflicting interpretation as artifact or real somatic variant. However, based on our approach and what is reported in the literature, we believe that in our cohort it is a true somatic variant. From a technical point of view, for library preparation, we used the Agilent SureSelect kit. Enrichment of the region of interest is based on probe capture by hybridization and the kit provides the proof-reading DNA polymerase Herculase II Fusion DNA Polymerase. NGS protocols for libraries preparation with these characteristics have been shown to overcome the issue of detection of this variant as an artifact (PMID: 30222780). Moreover, we applied an error suppression protocol, obtaining for this position very good sequencing parameters: a coverage of 957 reads with >10 paired-reads supporting the alternative allele. From a biological point of view, the ASXL1 p.G646Wfs*12 true somatic variant were usually age-related (PMID: 36652671) and in the 94% of cases were reported in combination with other mutations with a similar VAF (median 4 somatic mutations per case, range 1±8) (PMID: 30222780). Within our entire cohort of 130 individuals, we detected this mutation exclusively in this COVID-19 patient (ICU24), who is 68 years old and harbors together with this ASXL1 variant, other 6 variants with similar VAF. In particular, two of these variants are drivers and have been reported both in CHIP and myeloid malignancies: DMT3A p.R326H and SF3B1 p.R549C.

All other variants highlighted by the Reviewer, within our entire cohort of 130 individuals, are still found in only two patients and, interestingly, are identified only within the COVID-19 cohort, suggesting they could be specific variants for this clinical setting. All variants are well covered (mean coverage of 830 reads and mean of 20.5 reads supporting the alternative allele). According to our analytic workflow and variant filtering, we can unbiasedly remove these variants from our dataset. Finally, none of these variants is classified as driver or hazardous mutation, therefore, they do not impact the conclusions or the implications of our study.

2. What is the prevalence of CHIP (as defined by VAF >= 2%) in the cohorts? Without performing the analysis myself, it is not clear. The authors do state that "the vast majority of mutations...had a VAF <2%" but what about considering on a per-patient basis? I'm assuming the numbers will be too small for meaningful analysis of outcomes, but more information about traditional CHIP will allow a better comparison with previously published studies. Related to this, as compared to other studies (including Bolton et al.), the authors find here a higher proportion of TET2 variants (where Bolton and others have found more dominance of DNMT3A). Could this relate to the patient demographics, such as the number of patients with cardiovascular co-morbidity)? This may be worth discussing.

Response: We added the results restricting our analysis to CH variants with VAF≥2% in the Results section (line 171 line 176). Similar to what we observed considering all CH variants, we did not score statistically significant differences between COV-ICU patients and controls. These data show that restricting our analysis to CHIP mutations has no impact on the main results and conclusions of our study.

Concerning the number of mutations identified in DNMT3A or TET2, in the general cohort, we scored more mutations in DNMT3A compared to TET2, as reported in most studies. If the Reviewer is referring exclusively to the COVID-19 cohort, the difference we score is of 6 mutations in TET2 vs 5 mutations in DNMT3A. We think our numbers and the differences we scored are definitively too small to be able to observe significant correlations with any clinical or demographic parameters.

3. The lack of comparator for the 70s age group might be worth acknowledging. A recent study by Del Pozo Valero et al. found that myeloid-CH significantly increased risk of COVID-19 mortality among individuals ≥75 years, but not among those in their 60s (please see PMID: 36184726). Perhaps the lack of significance found here could be partly attributable to the relatively younger age groups that were compared. Please consider citing and discussing this study and potential limitation.

Response: We discuss the results of this recent paper in the DISCUSSION section, line 216-225.

4. The lack of impact of CH on clinical outcomes and parameters otherwise aligns with other studies, and the authors mention this in the manuscript. However, there may be other considerations regarding the statistically significant association of PD-CH in severe COVID-19. The authors mention "COVID-19 disease might instead select for hazardous CH-PD mutations". It might be pertinent to mention and consider the converse - that these types of PD-CH mutations could confer greater risk of severe disease, even if they're not directly impacting prognosis while in the ICU (i.e., greater implications on host antiviral defenses rather than prolonged illness).

Response: We added a comment in the DISCUSSION section (line 245-247)

5. Perhaps consider mentioning earlier in the METHODS, Patient cohort section, that patient demographic information (such as age, sex etc.) is available and point the reader to the appropriate tables.

Response: We added line 105 and 106 in the METHODS section with these pieces of information.

6. Regarding patient demographics, the supplemental table includes the exact date of birth and date of admission. This may be too identifying. The age (in years) at presentation may be sufficient, and the authors already state that patients presented/samples were collected in April 2020. Please consider if this type of (potentially identifying) information should be removed from the supplement.

Response: Thank you very much for highlighting this issue, we removed from S2 Table all potentially identifying data.

7. Gene names should be in italics (as an example, in the Introduction, line 59; but please check elsewhere).

Response: This has been fixed.

Reviewer #2: The authors re-evaluate the link between clonal hematopoiesis and COVID-19 severity by increasing the detection sensitivity of the defining somatic mutations (threshold 0.5%) in 24 patients with severe disease, 12 asymptomatic patients and 9 non-infected donors. The tested gene panel includes 80 genes, the coverage is 1350X, COSMIC and cBioportal are used to identify driver genes with a VAF that, in more than 80% of cases, was in the 0.5-2% range. The total number of detected mutations was similar in both group. Then, potential driver mutations and clonal complexity were analyzed, and these two parameters were higher in severe patients, without significant impact on clinical and biological parameters and disease outcome in this small cohort. Altogether, this study further argue for a limited impact of clonal hematopoiesis on severe COVID-19 outcome. The manuscript is clearly written and complements a series of previous analyses with various conclusions in small cohorts. A meta analysis will be needed for solid conclusions regarding the outcome. This report is a useful contribution to prepare this analysis.

In the introduction, the authors may stick to WHO 2022 classification of CHIP (Khoury et al, Leukemia 2022): CHIP refers to somatic mutations of myeloid malignancy-associated genes detected in the blood or bone marrow at a VAF ≥ 2% (≥4% for X-linked gene mutations in males) in individuals without a diagnosed hematologic disorder and without unexplained cytopenia.

Response: In the INTRODUCTION, line 51-55, we revisited the definition of CHIP according to the current WHO classification and cited the relevant publication, as suggested by the Reviewer.

The WHO classification also defines clonal cytopenia of undetermined significance (CCUS) as CHIP in the presence of one or more persistent cytopenias that are otherwise unexplained by hematologic or non-hematologic conditions and that do not meet diagnostic criteria for defined myeloid neoplasms. Did some of the patients included in this trial demonstrate a cytopenia before inclusion?

Response: None of the patients admitted to ICU had a positive clinical history for hematological disease and/or cytopenia. In the supplementary S2 Table, we report the results of the counts at admission to ICU of erythrocytes (RBC counts) and leucocytes (WBC counts). Counts are within the normal or normal-increased range for all patients.

The introduction also focuses on the cardiovascular risk of CHIP but many other risks have now been identified, the latest being liver diseases (see B Ebert's paper in Nature recently).

Response: We updated the INTRODUCTION (from line 67 to line 69) and cited the above paper within the REFERENCES.

Since they were included in 2020, do the authors have some information regarding the long-term outcome of the survivors?

Response: At one-year post-discharge from ICU, all patients included in the study were alive. We added a column reporting this piece of information in the S2 Table.

Attachment

Submitted filename: Response to Reviewers_PLOSone_Final.docx

Solimando Antonio Academic Editor

18 Sep 2023

PONE-D-23-04485R1High-sensitivity Analysis of Clonal Hematopoiesis Reveals Increased Clonal Complexity of Potential-Driver Mutations in Severe COVID-19 PatientsPLOS ONE

Dear Dr. Ronchini,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 02 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Antonio Solimando

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments :

The author should follow reviewer 1 indication and acknowledge in the final version of the manuscript the following limitations:

Small Sample Size: The study uses a relatively small sample size, which can limit the generalizability of the results. Further studies with larger patient cohorts should be undertaken to validate the findings.

Cross-Sectional Data: The study presents data captured at a single point in time, without analyzing the VAF variation over time. This cross-sectional approach might not capture the dynamic nature of clonal hematopoiesis and its implications in disease progression. Future studies should consider a longitudinal approach to track VAF variations over time to understand the role of these mutations in disease trajectory.

Age-Dependent Variation: While the study acknowledges age-dependent variations, it seems the age groups compared have different sample sizes, which might affect the statistical power of the analysis. Implementing age-matched control groups would strengthen the study.

Clinical Significance of CH-PD: The study identified a higher percentage of CH-PD mutations in the COV-ICU group compared to the control group; however, the clinical significance of these mutations, in terms of their impact on patient outcomes, remains unclear. The manuscript could benefit from a deeper analysis examining the clinical relevance of these findings, potentially linking it to patient outcomes or specific clinical characteristics.

Lack of Long-Term Follow-Up: The study does not follow up on the potential development of more serious conditions like MDS/AML based on the identified mutations, missing an opportunity to explore the long-term implications of the identified CH-PDs.

Implementations that will be needed in future studies (if beyond the scope of this manuscript):

Longitudinal Study: Implement a longitudinal study design to better assess the variations of VAF over time and potentially capture more nuanced relationships between CH, age, and disease severity.

Expanded Genetic Analysis: Consider an expanded analysis involving a greater number of genes to potentially uncover more associations between genetic variations and disease outcomes.

Multivariate Analysis: Incorporate multivariate analyses to control for potential confounding factors and to robustly examine the association between the identified mutations and disease outcomes.

Questions to be Addressed

VAF Variation over Time: The authors should discuss if and how VAF variation over time (months/years)can influence the outcomes and the observed CH. This can potentially be a marker for disease progression and should be considered in future studies.

Relation to CHIP/CCUS ICUS: It would be beneficial for the authors to discuss whether the allele frequencies observed can be a mirror of CHIP/CCUS ICUS or frankly MDS/AML in some or other cases. An acknowledgment of this potential association and a suggestion to explore this in future research would enrich the discussion.

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Reviewers' comments:

Reviewer's Responses to Questions

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the "Comments to the Author" section, enter your conflict of interest statement in the "Confidential to Editor" section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

***

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

***

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

***

4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

***

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

***

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank the authors for considering and addressing the majority of my comments. I am satisfied with the overall changes and believe these have improved the manuscript.

I do ask the authors to check the numbering of references and to ensure this has not been disrupted. This came to my attention when following references 30-32, the criteria for CH and PD-CH variants. In the original submission, these correctly referred to studies by Bolton et al., Young et al. and Acuna-Hidalgo et al. If I am not mistaken, the numbering of references has shifted in this revised version, and now 30-32 correspond to Abelson et al. (prediction of AML risk in CH), Fabre et al. (study of CH in twins), and Bolton et al. I do not believe this is correct. This problem may be more extensive than mentioned. Please review carefully reference numbers in text versus the reference list to ensure matching. Thank you.

Reviewer #2: (No Response)

***

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Reviewer #1: No

Reviewer #2: Yes: Eric Solary

***

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11 Oct 2023

We thank the Editor and the Reviewers for their comments. In the following pages are our point-by-point responses.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: we carefully reviewed our reference list and it appears complete and correct. By checking each journal website, none of the cited manuscript appears to be retracted.

Additional Editor Comments:

The author should follow reviewer 1 indication and acknowledge in the final version of the manuscript the following limitations:

Small Sample Size: The study uses a relatively small sample size, which can limit the generalizability of the results. Further studies with larger patient cohorts should be undertaken to validate the findings.

Response: We added a comment in the DISCUSSION section (line 298-301)

Cross-Sectional Data: The study presents data captured at a single point in time, without analyzing the VAF variation over time. This cross-sectional approach might not capture the dynamic nature of clonal hematopoiesis and its implications in disease progression. Future studies should consider a longitudinal approach to track VAF variations over time to understand the role of these mutations in disease trajectory.

Response: We added a comment in the DISCUSSION section (line 301-305)

Age-Dependent Variation: While the study acknowledges age-dependent variations, it seems the age groups compared have different sample sizes, which might affect the statistical power of the analysis. Implementing age-matched control groups would strengthen the study.

Response: One of the aims of our study was to analyze and compared samples from COV-ICU patients and controls that were collected during the same phase of the Covid-19 pandemic in the same regional area, in order to minimize the probability of infections from different viral variants, that clearly showed different microbiological characteristics and, more importantly, completely different clinical impacts on the infected individuals. Moreover, we wanted to analyze controls collected before the vaccination campaign, again, in order to avoid possible confounding effects on the hematopoietic cell system of our samples. In order to fulfill these requirements, for the controls, we took advantage of the study SOS-COV2 that included personnel working at our Institute and we analyzed all available samples collected from the oldest individuals of this cohort.

Clinical Significance of CH-PD: The study identified a higher percentage of CH-PD mutations in the COV-ICU group compared to the control group; however, the clinical significance of these mutations, in terms of their impact on patient outcomes, remains unclear. The manuscript could benefit from a deeper analysis examining the clinical relevance of these findings, potentially linking it to patient outcomes or specific clinical characteristics.

Response: In the manuscript all available clinical data, patient outcome and follow-up at one year for the COV-ICU cohort are included (S2 Table). As shown in S4 Figure entitled: "Survival and clinical parameters in COV-ICU patients", comparing COV-ICU patients with CH-PD mutations versus patients without CH-PD mutations, we did not identify statistically significant differences. This is acknowledged in the Results section (line 201-203). We added the reference to S2 Table and the no significance of data.

Lack of Long-Term Follow-Up: The study does not follow up on the potential development of more serious conditions like MDS/AML based on the identified mutations, missing an opportunity to explore the long-term implications of the identified CH-PDs.

Response: The absolute risk of hematopoietic malignancy development in persons with CH is low and individuals with CH progress to malignancy at a rate of about 0.5 to 1% per year. It has been reported that 4% of persons with CH develop hematopoietic malignancies in 8 years (Jaiswal et al., NEJM 2014). In the study from Abelson et al. (Nature 2018) individuals with CH developed myeloid neoplasms with a median follow-up of 7.6 years. Moreover, quantitatively the authors found that CH-PD mutations conferred around a 2-fold increase risk of developing AML per 5% increase in clone size. Moreover, CH parameters that increase the risk of leukemia transformation are linked to the presence of: clones with VAF>10%, more than 1 mutated gene, mutations in specific driver genes and specific aminoacid changes. Considering that: i) our cohort is small, ii) the COV-ICU patients had normal hematological counts and iii) in our COV-ICU patients the majority of clones have VAF<2%, we believe that the probability of scoring the development of myeloid neoplasms in the time frame of follow-up of 3 is extremely low. Moreover, although very interesting and important, this is beyond the scope of our study, which was the assessment of CH at high sensitivity in the two cohorts.

Implementations that will be needed in future studies (if beyond the scope of this manuscript):

Longitudinal Study: Implement a longitudinal study design to better assess the variations of VAF over time and potentially capture more nuanced relationships between CH, age, and disease severity.

Expanded Genetic Analysis: Consider an expanded analysis involving a greater number of genes to potentially uncover more associations between genetic variations and disease outcomes.

Multivariate Analysis: Incorporate multivariate analyses to control for potential confounding factors and to robustly examine the association between the identified mutations and disease outcomes.

Response: We agree with the editor that all these issues are very insightful. However, they would require the design and development of new specific trials and tools that are beyond the scope of this manuscript.

Questions to be Addressed

VAF Variation over Time: The authors should discuss if and how VAF variation over time (months/years)can influence the outcomes and the observed CH. This can potentially be a marker for disease progression and should be considered in future studies.

Response: We added a comment in the DISCUSSION section (line 236-250)

Relation to CHIP/CCUS ICUS: It would be beneficial for the authors to discuss whether the allele frequencies observed can be a mirror of CHIP/CCUS ICUS or frankly MDS/AML in some or other cases. An acknowledgment of this potential association and a suggestion to explore this in future research would enrich the discussion.

Response: Most of the genomic studies on myeloid neoplasms are based on whole exome or large gene panels sequencing analysis and usually, consistent with normal clinical practice, use VAF threshold of 1% for hotspot mutations and of 5% for variants of unknown significance. Moreover, in frank myeloid neoplasms usually important clonal expansions has already taken place. It is, therefore, not surprising that most of the mutations in common with our study display higher VAF in dataset of myeloid neoplasms or CHIP compared to the one we scored. Nonetheless, we found similar VAF in a few patients with myeloid neoplasms and we added a comment addressing this issue in the DISCUSSION section (line 256-268).

Reviewer #1: I thank the authors for considering and addressing the majority of my comments. I am satisfied with the overall changes and believe these have improved the manuscript.

I do ask the authors to check the numbering of references and to ensure this has not been disrupted. This came to my attention when following references 30-32, the criteria for CH and PD-CH variants. In the original submission, these correctly referred to studies by Bolton et al., Young et al. and Acuna-Hidalgo et al. If I am not mistaken, the numbering of references has shifted in this revised version, and now 30-32 correspond to Abelson et al. (prediction of AML risk in CH), Fabre et al. (study of CH in twins), and Bolton et al. I do not believe this is correct. This problem may be more extensive than mentioned. Please review carefully reference numbers in text versus the reference list to ensure matching. Thank you.

Response: The numbering of references changed from the original manuscript because, as mentioned in the Rebuttal letter for the first round of revision, we added new references, according to the suggestions by the Reviewers. We carefully checked the numbering reported in the revised manuscript and it is correct. Indeed, for annotation of CH variants as potential drivers, we used the definitions and the parameters reported in Bolton et al., Abelson et al. and Fabre et al. In particular, we used as reference the Supplemental Table 2 published in Fabre et al. plus all parameters described in the other manuscripts. To the best of our knowledge, there is no a formal definition of CH-Driver mutations in Young et al. or Acuna-Hidalgo et al.

Attachment

Submitted filename: Ronchini_ResponseToReviewers_PONE-D-23-04485_R1.docx

Solimando Antonio Academic Editor

7 Nov 2023

PONE-D-23-04485R2High-sensitivity Analysis of Clonal Hematopoiesis Reveals Increased Clonal Complexity of Potential-Driver Mutations in Severe COVID-19 PatientsPLOS ONE

Dear Dr. Ronchini,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Antonio Solimando

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Overall, the authors have made efforts to address the reviewers' comments and justify any limitations or decisions made in the study, while also updating the reference list and clarifying any confusion around it. They are open about the limitations of their study and have incorporated suggestions from the reviewers into the revised manuscript where possible. They also highlight areas that could be explored in future research but which were beyond the scope of their current study. Indeed, the authors' responses in the rebuttal letter generally address the concerns raised by the reviewers. However, there are always areas for improvement, both in the manuscript and in the way the authors communicate their revisions to the reviewers and the readers. Here are some suggestions:

Clarification on Reference Numbering:

The response about the reference numbering was somewhat unclear. The authors should make sure that the reference numbers in the text match the updated reference list. They should explicitly confirm that they have double-checked the in-text citations against the updated reference list to ensure all references are accurately represented.

Explicit Acknowledgement of Limitations:

While the authors have acknowledged the limitations pointed out by the reviewers in the discussion section, it may be beneficial to include a succinct summary of these limitations in the abstract or conclusions to ensure readers are immediately aware of the study's scope and constraints.

Methodological Rigor and Follow-Up:

Although the authors mention that long-term follow-up and expanded studies are beyond the scope of the current study, they could outline a more detailed plan for future research. This might include potential study designs, collaborations, or funding opportunities they plan to seek in order to address these limitations.

Detailed Justification for Methodological Choices:

For the controls chosen from the SOS-COV2 study, the authors should provide a more detailed rationale for why these specific samples were selected, beyond the timing of collection and the pre-vaccination status, such as demographic similarities or exposure risks that match the study group.

Statistical Considerations:

The authors should consider discussing any additional statistical tests or models that could be applied to their data in future studies to account for confounding variables not addressed in this study, such as multivariate regression analyses or propensity score matching.

Potential Implications and Applications:

A deeper exploration of how the identified mutations could be monitored or used in clinical practice might add value to the discussion, even if this application is not immediately feasible.

Accessibility of Data and Materials:

Ensuring that all supplementary materials and methods are easily accessible to readers and researchers for reproducibility purposes is important. If the authors have not already done so, they might consider including a statement confirming the availability of such resources or outlining how they can be accessed.

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10 Nov 2023

In the following pages are our point-by-point responses.

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: we double-checked the in-text citations against the updated reference list and ensured that all references are accurately represented. By checking each journal website, none of the cited manuscript appears to be retracted.

Additional Editor Comments:

Overall, the authors have made efforts to address the reviewers' comments and justify any limitations or decisions made in the study, while also updating the reference list and clarifying any confusion around it. They are open about the limitations of their study and have incorporated suggestions from the reviewers into the revised manuscript where possible. They also highlight areas that could be explored in future research but which were beyond the scope of their current study. Indeed, the authors' responses in the rebuttal letter generally address the concerns raised by the reviewers. However, there are always areas for improvement, both in the manuscript and in the way the authors communicate their revisions to the reviewers and the readers. Here are some suggestions:

Clarification on Reference Numbering:

The response about the reference numbering was somewhat unclear. The authors should make sure that the reference numbers in the text match the updated reference list. They should explicitly confirm that they have double-checked the in-text citations against the updated reference list to ensure all references are accurately represented.

Response: we confirm that we double-checked the in-text citations against the updated reference list and ensured that all references are accurately represented.

Explicit Acknowledgement of Limitations:

While the authors have acknowledged the limitations pointed out by the reviewers in the discussion section, it may be beneficial to include a succinct summary of these limitations in the abstract or conclusions to ensure readers are immediately aware of the study's scope and constraints.

Response: we acknowledged the limitations of our study in the abstract (line 49-53).

Methodological Rigor and Follow-Up:

Although the authors mention that long-term follow-up and expanded studies are beyond the scope of the current study, they could outline a more detailed plan for future research. This might include potential study designs, collaborations, or funding opportunities they plan to seek in order to address these limitations.

Response: A possible strategy would be to retrospectively retrieve DNA from PB that have been collected from COVID19 patients treated in ICU and, then, longitudinally at different follow-up post-discharge, in order to perform our high sensitivity CH analysis. However, we really believe this is beyond the scope of the current study.

Detailed Justification for Methodological Choices:

For the controls chosen from the SOS-COV2 study, the authors should provide a more detailed rationale for why these specific samples were selected, beyond the timing of collection and the pre-vaccination status, such as demographic similarities or exposure risks that match the study group.

Response: we added these details in the Materials and Methods section (line 110-118).

Statistical Considerations:

The authors should consider discussing any additional statistical tests or models that could be applied to their data in future studies to account for confounding variables not addressed in this study, such as multivariate regression analyses or propensity score matching.

Response: Increasing the number of patients analyzed, one could apply machine learning models to discriminate between ICU patients and Controls by integrating multivariate clinical features with our genomic features. However, we believe it is premature to suggest any additional specific type of statistical tests or models without additional available information.

Potential Implications and Applications:

A deeper exploration of how the identified mutations could be monitored or used in clinical practice might add value to the discussion, even if this application is not immediately feasible.

Response: We applied a high sensitivity NGS technology which requires extensive experimental and bioinformatic workflows, is expensive, and is not easily implementable in clinical practice. To our knowledge, up to day, the only alternative available technique with similar sensitivity is digital PCR, not used in standard clinical practice. Moreover, how to counsel people with CH or monitor them prospectively is an area of uncertainty and a quite challenging task at the moment. Indeed, there are no clinical indications or guidelines on how to manage the presence of CH mutations nor there are established interventions to try to eradicate CH clones. We believe it is really too early to include this information in our paper, especially, considering that we mainly detected low frequency mutations, which require high sensitivity techniques to be monitored.

Accessibility of Data and Materials:

Ensuring that all supplementary materials and methods are easily accessible to readers and researchers for reproducibility purposes is important. If the authors have not already done so, they might consider including a statement confirming the availability of such resources or outlining how they can be accessed.

Response: we added a Data Availability Statement (line 322-324).

Attachment

Submitted filename: Ronchini_ResponseToReviewers_PONE-D-23-04485R2.docx

Solimando Antonio Academic Editor

28 Nov 2023

High-sensitivity Analysis of Clonal Hematopoiesis Reveals Increased Clonal Complexity of Potential-Driver Mutations in Severe COVID-19 Patients

PONE-D-23-04485R3

Dear Dr. Ronchini,

We're pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you'll receive an e-mail detailing the required amendments. When these have been addressed, you'll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Antonio Solimando

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Solimando Antonio Academic Editor

28 Dec 2023

PONE-D-23-04485R3

PLOS ONE

Dear Dr. Ronchini,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Antonio Solimando

Academic Editor

PLOS ONE

We thank IEO Covid Team and all IEO personnel that participated to sample collections and laboratory measurements for the SOS-COV2 study in IEO and the Genomics Unit of the Department of Experimental Oncology of IEO for sequencing of the CH libraries.