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. 2025 Feb 25;20(2):e0319194. doi: 10.1371/journal.pone.0319194

The role of renal and liver function in clinical ctDNA testing

Jens Bo Koudahl Conrad 1,2, Tenna Vesterman Henriksen 1,2, Jesper Berg Nors 1,2,3, Mads Heilskov Rasmussen 1,2, Mai-Britt Worm Ørntoft 1,2,4, Nis Hallundbæk Schlesinger 5, Per Vadgaard Andersen 6, Kåre Andersson Gotschalck 2,7, Claus Lindbjerg Andersen 1,2,*
Editor: Elingarami Sauli8
PMCID: PMC11856342  PMID: 39999130

Abstract

Circulating tumor DNA (ctDNA) has high clinical potential in early cancer detection. The renal system and the liver are involved in clearing circulating cell free DNA (cfDNA) from the blood. Recent studies on mice show that inhibiting the liver’s ability to clear cfDNA results in elevated ctDNA levels in blood samples. Emphasizing the need for studies in humans exploring if markers of renal and liver function are associated with cfDNA and ctDNA levels in the blood. The present study investigates if cfDNA level, ctDNA level and ctDNA detection is affected in colorectal cancer (CRC) patients with clinical biomarkers indicative of low renal and liver function. We requisitioned standard laboratory tests of renal and liver function, measured within thirty days of curative intended surgery from 846 stage I-III CRC patients. For each patient, matching preoperative cfDNA and ctDNA data was available. We investigated the correlation between impaired renal and liver function and cfDNA level, ctDNA level, and ctDNA detection. The findings revealed that variation in renal and liver function in stage I-III CRC patients did not affect cfDNA level, ctDNA level, or ctDNA detection and that ctDNA test results remain stable over a wide range of renal and liver biomarker results.

1. Introduction

Measurements of circulating tumor DNA (ctDNA) is an emerging tool for early cancer detection, allowing for in-depth monitoring of treatment effect and disease progression [1–5]. Despite ctDNA detection technologies becoming increasingly sensitive, ctDNA is not detectable in all cancer patients. The ability to detect ctDNA is dependent on the level of ctDNA in the blood, which again is determined by the release and clearance-rate of tumor DNA [6]. The supply of ctDNA to the blood is driven by cancer cell turnover [7], which is closely linked to disease stage and location [8]. In healthy individuals, the half-life of ctDNA in the circulation is estimated to ~ 2 hours [8]. Circulating cell-free DNA (cfDNA) in blood has been reported to be cleared via the liver and kidneys [9]. In support of this, increased cfDNA levels have been reported in patients with poor renal [10–12] and liver function [13,14]. Furthermore, it was recently reported that transiently blocking the liver cfDNA clearance function in mice increased the cfDNA level in circulation by up to 10-fold, and that this in turn increased the sensitivity for detecting ctDNA in mice with small tumors [15].

Patients afflicted with cancer disproportionately suffer from liver and renal diseases when compared to the background population. In patients with non-alcoholic fatty liver disease the risk of colorectal cancer (CRC) is elevated 3-fold [16] and in patients with CRC abnormally low renal function has an estimated prevalence of over 70% [17]. Furthermore, the presence of a cancer in the body may in and of itself degrade the condition and function of the liver and kidneys [18–20]. Hence, it may be hypothesized that CRC patients affected by poor liver and renal function may have attenuated ctDNA clearance, and therefore augmented levels of ctDNA. Potentially, ctDNA testing will be more sensitive in these patients than in patients without liver and kidney comorbidities.

CRC patients in Denmark undergo a range of laboratory tests prior to curatively intended surgery. These include creatinine, glomerular filtration rate (eGFR), sodium, and potassium. Collectively, these parameters estimate the fluid status and kidney function of a patient; Indeed, estimates of eGFR form the foundation for diagnosing patients with chronic kidney disease. Liver function is estimated via blood tests of bilirubin, alkaline phosphatase, and alanine transaminase among others. The laboratory test data is archived and readily available, providing a detailed view into the renal and liver function of CRC patients in the Danish healthcare system. To investigate the impact of renal and liver function on cfDNA level, ctDNA level, and ctDNA detection probability, we analyzed a cohort of CRC patients for which preoperative liver and renal function laboratory tests as well as information of cfDNA and ctDNA level was available.

2. Methods

2.1. The patient cohort

The cohort for this study comprised consecutively recruited patients planned to receive a curative intended surgical resection of stage I-III CRC. All patients were diagnosed and treated in accordance with Danish guidelines (DCCG.dk). The participants were recruited from 2012 to 2022 at nine hospitals in the Capital, Central and Southern Denmark regions. As part of the study, patients had blood collected prior to and after surgery with the aim to retrospectively measure cfDNA and ctDNA. All patients gave written informed consent in accordance with the World Medical Association Declaration of Helsinki. The project was approved by The Central Denmark Committees on Health Research Ethics (case j. no. 1-10-72-3-18 and j. no. 1-10-72-223-14).

2.2. Extracting data on laboratory test results

Laboratory test results of renal and liver function were requisitioned from the local “Clinical Laboratory Information System Research Databases” (LABKA) in each of the involved Danish regions. To gauge liver function, alkaline phosphatase, bilirubin and alanine transaminase results were requisitioned. To gauge renal function, creatinine, eGFR, sodium, and potassium results were chosen. The LABKA databases do not report precise eGFR results above 90 mL/min/1.73m2. Therefore, any eGFR test result of > 90 mL/min/1.73m2 was registered as 90 mL/min/1.73m2 in this study.

2.3. Classification of laboratory test results

The laboratory results were categorized according to the reference range for each sample. In Denmark, the hospital system is organized in five independent regions, each of which define the normal reference range for their laboratories. The reference range for a specific patient’s test result was defined using the reference range specific to the Region the patient was recruited from, accounting for age and sex where applicable [21–23] (S1 Table in S1 Text). Based on the reference ranges the results for each patient were categorized as “Below reference”, “Within reference”, or “Above reference”.

2.4. Tumor and cfDNA

The ctDNA quantification data included in this paper have been previously published in their own right [24]. Detailed methodology is described there and in the associated methodology papers (24-28). In brief, whole-exome sequencing was conducted on paired tumor and buffy coat DNA samples from each patient. This data was evaluated for clonal mutations, selected based on variant allele frequencies and estimates of cancer cell purity, tumor ploidy, and allele-specific copy numbers [25]. Based on the available clonal mutations, ctDNA analysis was conducted using either droplet digital PCR targeting a single, clonal, small-nucleotide variant [26,27] or deep targeted panel sequencing of 12 genes frequently mutated in CRC [28].

2.5. Pairing biomarkers of renal and liver function to ctDNA test results

Laboratory test results were matched with the preoperative and postoperative cfDNA samples. For patients with repeated laboratory measurements, the measurement closest in time to the cfDNA blood collection was chosen. Only test results from within 30 days before the operation were considered when matching results to preoperative ctDNA and cfDNA measurements.

2.6. Defining low renal function

In this study, we define a 30-day preoperative eGFR as the mean of all eGFR measurements conducted in the month leading up to the operation. The formulas used by the regional hospitals to calculate eGFR is available in S1 File. The median number of eGFR measurements per patient in the month up to operation was 2 (IQR 1-3). The 30-day preoperative eGFR was used to define whether a patient had low renal function or normal renal function. In accordance with Kidney Disease: Improving Global Outcomes (KDGIO) guidelines (https://kdigo.org) patients were categorized as having low renal function if their average eGFR was below 60 mL/min/1.73m2

2.7. Statistical analysis

The ctDNA status (detected or undetected), ctDNA level, and cfDNA level were the dependent variables whereas the laboratory test results were the primary explanatory variables. To investigate the relationship between the liver function and the risk of a positive ctDNA call, binomial logistic regression analysis was applied. The association between renal function and ctDNA test results was similarly analyzed with a binomial logistic regression. A log-log regression was used to analyze the relationship between the liver/renal laboratory test level and the ctDNA or cfDNA level. Only ctDNA positive patients were included in the log-log regression analysis. All regression models were adjusted for patient age at date of operation and the pT and pN categories of the tumor. In all analyses, the false discovery rate was controlled using the Benjamini-Hochberg method. A significant result was defined as p <  0.05 after Benjamini-Hochberg adjustment. All statistical analysis was conducted using the programming language R, version 4.3.1.

3. Results

In total, we included 846 stage I-III CRC patients. Patient characteristics are presented in Table 1. We measured cfDNA and ctDNA preoperatively on all patients. Clinical routine biomarker levels indicative of renal function and liver function were available on different subsets of patients (S2 Table in S1 Text).

Table 1. Clinical characteristics of the study cohort.

Characteristic N =  8461
Age 71 (64, 77)
Sex
 Female 383 (45%)
 Male 463 (55%)
T stage
 pT1 35 (4.1%)
 pT2 90 (11%)
 pT3 635 (75%)
 pT4 86 (10%)
N stage
 pN0 569 (67%)
 pN1 180 (21%)
 pN1c 6 (0.7%)
 pN2 91 (11%)
Location
 Left side 420 (50%)
 Right side 387 (46%)
 Rectum 39 (4.6%)
1Median (IQR); n (%)
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3.1. Association between renal function and cfDNA and ctDNA

To explore the impact of renal function on ctDNA and cfDNA test results, we investigated whether the sodium, potassium, creatinine, and eGFR levels were correlated to cfDNA level, ctDNA level, and ctDNA detection status. None of the renal biomarkers were significantly associated with cfDNA level, ctDNA level, or ctDNA detection status (Fig 1). Full regression results are available in S3–S6 Tables in S1 Text.

Fig 1. Association between liver function tests and the cfDNA level, ctDNA level, and ctDNA detection.

Fig 1

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(A) Scatterplots comparing cfDNA level (top) and ctDNA level (bottom) to renal function test results. (B) Scatterplots comparing ctDNA level to renal function test results. In A and B dots are colored for the reference range categories. A log-log regression was used to analyze the relationship between the laboratory test results and the ctDNA or cfDNA level. (C) Box plots depicting test results in ctDNA negative and ctDNA positive patients. The relationship between the laboratory test result and the risk of a positive ctDNA call was analyzed with a binomial logistic regression. (D) Stacked bar chart showing the proportion of patients in each reference range category in ctDNA negative and ctDNA positive patients. For eGFR, a result above 90 mL/min/1.73m2 was treated as 90 mL/min/1.73m2. All regression models were adjusted for patient age at date of ctDNA sampling, and the pT and pN categories of the tumor. In all analyses, the false discovery rate was controlled using the Benjamini-Hochberg method. A significant result was defined as p <  0.05 after Benjamini-Hochberg adjustment. Note that different patients can have different reference ranges depending on age, sex, and region of origin. Therefore, the same laboratory result can be categorized in different reference range categories.

We further investigated whether patients with normal and low renal function differed in their cfDNA and ctDNA levels (Fig 2). A correlation between increased cfDNA level and low renal function (OR =  2.415 p =  0.007) was observed. However, after adjusting for patient age and disease stage the association disappeared (OR =  1.380 p = 0.369) (Fig 2A). No association was observed between ctDNA level and renal function neither with or without adjustments for age and low renal function were observed (Fig 2B). Full regression results are available in S7 Table in S1 Text.

Fig 2. Box plot showing the cfDNA level (A) and ctDNA level (B) in patients with low renal function and patients with normal renal function.

Fig 2

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The association was assessed using binomial logistic regression. The statistical analysis was adjusted for pT stage and pN stage as well as age at sample date.

3.2. Association between liver function and cfDNA and ctDNA

To explore the possible link between liver function and ctDNA and cfDNA levels, we analyzed whether the bilirubin, alanine transaminase, and alkaline phosphatase were correlated to cfDNA level, ctDNA level, and ctDNA detection rate (Fig 3). We found that elevated bilirubin levels were correlated with increased cfDNA levels (R =  0.148, p =  0.022). However, there were no statistically significant correlations to ctDNA level or ctDNA detection status. Neither alkaline phosphatase nor alanine transaminase were significantly associated with cfDNA and ctDNA levels nor with ctDNA detection status. Full regression results are available in S3–S6 Tables in S1 Text.

Fig 3. Association between liver function tests and the cfDNA level, ctDNA level, and ctDNA detection.

Fig 3

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(A) Scatterplots comparing cfDNA level (top) and ctDNA level (bottom) to liver function test results. (B) Scatterplots comparing ctDNA level to liver function test results. In A and B dots are colored for the reference range categories. A log-log regression was used to analyze the relationship between the laboratory test results and the ctDNA or cfDNA level. (C) Box plots depicting test results in ctDNA negative and ctDNA positive patients. The relationship between the laboratory test result and the risk of a positive ctDNA call was analyzed with a binomial logistic regression. (D) Stacked bar chart showing the proportion of patients in each reference range category in ctDNA negative and ctDNA positive patients. All regression models were adjusted for patient age at date of ctDNA sampling, and the pT and pN categories of the tumor. In all analyses, the false discovery rate was controlled using the Benjamini-Hochberg method. A significant result was defined as p <  0.05 after Benjamini-Hochberg adjustment. Note that different patients can have different reference ranges depending on age, sex, and region of origin. Therefore, the same laboratory result can be categorized in different reference range categories.

4. Discussion

In this study, we investigated whether preoperative laboratory biomarkers of renal and liver function were correlated to preoperative cfDNA level, ctDNA level, and ctDNA detection in CRC patients. We paired cfDNA test results to data from the Danish healthcare system’s LABKA databases.

We observed that patients with higher bilirubin levels tended to have higher levels of cfDNA, though the association was minor. However, the increased cfDNA levels did not translate to increased ctDNA levels or ctDNA detection rates; if increased bilirubin levels were indicative of decreased cfDNA clearance, we would expect the ctDNA level to increase as well. None of the other investigated markers showed statistically significant associations with cfDNA or ctDNA test results. We did find that patients with low renal function had increased levels of cfDNA, but this association lost statistical significance when the analysis was adjusted for patient age. This likely reflects that advanced age both associated with increased cfDNA levels and lower renal function [29,30]. In addition we conducted a supplementary investigation of cfDNA levels in the postoperative setting (S2 File) and found no significant association between postoperative cfDNA level and biomarkers of renal and liver function.

Our results suggest that CRC patients eligible for curative intended surgery do not experience abnormal ctDNA and cfDNA clearance based on their estimated renal and liver function. The vast majority of the standard renal and liver marker laboratory test results were within the normal reference ranges. One can expect that an effect on cfDNA clearance would be more apparent in patients with severe liver disease and end-stage renal disease. Indeed, in patients with nonalcoholic fatty liver disease, Karlas et al. found that disease severity correlated with higher cfDNA levels [14]. Additionally, Coimbra et al. found that cfDNA was elevated in patients who suffered from end-stage renal disease [11]. It may therefore be that the liver and renal function of the included CRC patients is not low enough to affect ctDNA clearance. Patients with much lower renal and liver function, than those included in this study, can be found in Denmark, but these patients would very rarely be candidates for curative intended surgery in the Danish healthcare system.

Although our results indicate that ctDNA clearance inhibition does not occur naturally in stage I-III CRC patients, Martin-Alonso et al. [15] found that blockage of liver-mediated cfDNA clearance in mice resulted in increased ctDNA concentrations. If also effective in humans, this could have great implications for future ctDNA testing, as low ctDNA concentrations is the most challenging aspect of ctDNA detection. However, if liver-based ctDNA clearance was already inhibited in a subset of CRC patients, the ctDNA priming strategy based on inhibiting liver clearance might have limited effect. Our observation that ctDNA levels are stable across the range of liver biomarkers indicate that this should not be a concern.

While ctDNA detection is primarily utilized as a strong predictor of cancer relapse, ctDNA level in the blood and its growth rate also has utility as a prognostic marker [27]. The variable ctDNA levels detected in blood samples remains a challenge for the field though [8]. Improving our understanding of what factors determine ctDNA level in patients will allow researchers to better account for biological variation when they use ctDNA level as a prognostic indicator. Indeed, much work is being done identifying which patient parameters influence ctDNA availability [31–33].

While using hospital databases to gather data on blood-based clinical biomarkers allowed for a highly representative dataset on a large cohort of CRC patients, it comes with some limitations. Most of the clinical biomarkers were collected on the discretion of attending clinicians. The availability of some biomarkers will therefore be subject to confounding by indication. Additionally, when using the laboratory databases, analyses of continuous measures were limited for markers with values below or above the quantifiable range. Our study focused on laboratory test results in the short 30-day period before curative intended surgery as this period is the standard length of the Danish preoperative CRC cancer work-up program. However, this has the limitation of not meeting KDGIOs recommended 3-month sampling period for assessing long-term renal function. While most of our patients had multiple eGFR measurements in the 30-day period, a minor fraction had just a single eGFR measurement (n =  181 37.6%). These singular measurements provide important information, yet will not account for the patient’s biological variance in eGFR. Finally, ctDNA samples and clinical biomarker samples were not always collected on the same day, which may introduce some variance.

In summary, this study indicates that variation in liver and renal function among patients with localized CRC does not influence ctDNA level or ctDNA detection. While more research is required to elucidate factors governing ctDNA release and clearance, implementation of ctDNA priming strategies in a human setting could potentially increase ctDNA levels, thus radically increasing ctDNA test sensitivity. For now, it is encouraging that abnormal liver and renal function does not need to be considered when conducting ctDNA testing, as the ctDNA clearance can be considered robust across a wide range of test results in CRC patients undergoing curative intended treatment.

Supporting Information

S1 Fig. Association of eGFR with cfDNA level stratified by age.

Figures showing grouped eGFR levels and continuous cfDNA levels stratified for patient age. Associations were calculated with a paired Wilcoxon signed rank test.

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pone.0319194.s001.eps (874.5KB, eps)
S2 Fig. Association of eGFR with ctDNA level stratified by age.

Figures showing grouped eGFR levels and continuous ctDNA levels stratified for patient age. Associations were calculated with a paired Wilcoxon signed rank test.

(EPS)

pone.0319194.s002.eps (821.9KB, eps)
S1 Text

S1 Table. Reference ranges for the laboratory tests. S2 Table. Number of patients for whom a specific laboratory test was available. S3 Table. Full log-log linear regression results for cfDNA. S4 Table. Full log-log linear regression results for ctDNA. S5 Table. Full binomial logistic regression results for ctDNA detection compared to laboratory test result. S6 Table. Full binomial logistic regression results for ctDNA detection compared to reference range category of laboratory test. S7 Table. Full binomial logistic regression results for ctDNA and cfDNA compared to low and high renal function.

(XLSX)

pone.0319194.s003.xlsx (42.5KB, xlsx)
S1 File. Description of eGFR calculation formulas.

(DOCX)

pone.0319194.s004.docx (19.6KB, docx)
S2 File. Postoperative analysis of cfDNA and laboratory biomarkers of renal and liver function.

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pone.0319194.s005.docx (41.9KB, docx)
S3 Fig. Potassium_chart.

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pone.0319194.s006.eps (220.7KB, eps)
S4 Fig. Postop_cohort_funnel.

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pone.0319194.s007.eps (135.1KB, eps)
S5 Fig. Postop_cfDNA_linear_regression.

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pone.0319194.s008.eps (489.9KB, eps)
S6 Fig. Postop_renal_function.

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pone.0319194.s009.eps (80.1KB, eps)

Acknowledgments

We thank the blood donors, CRC patients, and The Danish Cancer Biobank for contributing clinical material. The study was conducted as part of the Danish National Center for Circulating Tumor DNA Guided Cancer Treatment.

Data Availability

Because the data contains sensitive personal patient information, it cannot be made publicly available. Access to data requires that the data requestor (legal entity) enter into Collaboration and Data Processing Agreements, with the Central Denmark Region (the legal entity controlling and responsible for the data). Inquiries for access can be addressed to the Data Access Committee at Department of Molecular Medicine, Aarhus University Hospital (contact via moma@rm.dk).

Funding Statement

This study was funded by a scholar-stipend from the Danish Independent Research Fund (Conrad) and support from the Novo Nordisk Foundation [grant number NNF22OC0074415 (Andersen)], Innovation Fund Denmark [grant number 9068-00046B (Andersen)] and the Danish Cancer Society [grant numbers R231-A13845, R257-A14700, R352-A20664 (Andersen)]. The funding organizations and entities listed above had no influence on the study’s design, data collection, analysis, interpretation of the data, manuscript preparation and review, or decision to submit for publication.

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Decision Letter 0

Elingarami Sauli

17 Sep 2024

PONE-D-24-32619The role of renal and liver function in clinical ctDNA testingPLOS ONE

Dear Dr. Andersen,

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.

==============================The authors need to improve and detail the methodology, including patients selection.

==============================

Please submit your revised manuscript by Nov 01 2024 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Elingarami Sauli, PhD

Academic Editor

PLOS ONE

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: No

Reviewer #2: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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

**********

5. 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: The authors investigated whether cfDNA and ctDNA levels are influenced by impaired renal or liver function in patients undergoing curative-intent surgery for early-stage colorectal cancer. Their findings suggest that cfDNA and ctDNA levels remain stable across a broad spectrum of renal and liver function metrics.

However, several important limitations should be considered:

1. The study is focused on pre-operative cfDNA/ctDNA levels, but in clinical practice, post-operative ctDNA is more relevant. It cannot be assumed that post-operative cfDNA/ctDNA levels would remain unaffected by varying degrees of renal or liver function, particularly given the additional variable of DNA fragments released during surgery, which could influence ctDNA clearance in patients with impaired renal or liver function.

2. The method used to calculate eGFR is not clearly described, raising concerns about the accuracy and reliability of the renal function assessment.

3. The manuscript does not clarify whether patients had chronic kidney disease, defined as kidney damage or decreased kidney function persisting for three or more months. A single instance of slightly elevated creatinine does not accurately reflect true renal function.

4. Figure 1 indicates that only a small number of patients had creatinine levels above the normal reference range or an eGFR below the normal reference range. Drawing broad conclusions from such limited data may not be justified. The same concern applies to liver function tests, particularly serum bilirubin levels.

5. It would be beneficial to categorize patients with eGFR below 60 into subgroups based on the severity of renal impairment (e.g., eGFR 45-59, 30-44, etc.) to determine whether ctDNA levels are affected at specific eGFR threshold.

In summary, this manuscript does not offer any clinically valuable information.

Reviewer #2: The manuscript reports an interesting concept of impact of variation in renal and liver functions on the detection of ctDNA/cfDNA in patients with resectable colorectal cancer. The conclusion is that there is no such effect. I have the following comments.

1. The only major limitation in this study is that the majority of the participants have their renal/liver functions within reference range and hardly any participant has severe renal or liver dysfunction. This should be clearly highlighted as a limitation and reason for contradiction from the the pre-clinical and previously published reports.

2. It appears the bloods samples for ctDNA were taken both pre and post-surgery, but only pre-op data is presented. Did they compare post op ctDNA with post-op renal/liver functions?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2025 Feb 25;20(2):e0319194. doi: 10.1371/journal.pone.0319194.r003

Author response to Decision Letter 0

9 Oct 2024

Reviewer #1: The authors investigated whether cfDNA and ctDNA levels are influenced by impaired renal or liver function in patients undergoing curative-intent surgery for early-stage colorectal cancer. Their findings suggest that cfDNA and ctDNA levels remain stable across a broad spectrum of renal and liver function metrics.

However, several important limitations should be considered:

1. The study is focused on pre-operative cfDNA/ctDNA levels, but in clinical practice, post-operative ctDNA is more relevant. It cannot be assumed that post-operative cfDNA/ctDNA levels would remain unaffected by varying degrees of renal or liver function, particularly given the additional variable of DNA fragments released during surgery, which could influence ctDNA clearance in patients with impaired renal or liver function.

Author Response: We appreciate this input from the reviewer. We agree that it is not possible to conclude that post-operative ctDNA levels are unaffected by the liver and renal function of the patients in this cohort. However, we believe that the complexity of the postoperative treatment regime makes it infeasible to draw conclusions about postoperative ctDNA levels with registry data. See our answer to comment number 2 from reviewer #2 for additional details.

2. The method used to calculate eGFR is not clearly described, raising concerns about the accuracy and reliability of the renal function assessment.

Author Response: We are thankful to the reviewer for this vigilant observation. The eGFR results were requisitioned from the regional databases of hospital laboratories using nationally standardized testing methodology. We have added a translated description of the eGFR assessment methodology and eGFR calculation formula in supporting information and revised the manuscript to refer to this [line 108. S1 File].

“In this study, we define a 30-day preoperative eGFR as the mean of all eGFR measurements conducted in the month leading up to the operation. The formulas used by the regional hospitals to calculate eGFR is available in S1 File.”

For ease of the reviewer we have also added them here:

Female

P-Creatinine ≤62 µmol/l:

eGFR = 144 x (P-Creatinine /(0.7 x 88.4))-0.329 x 0.993^age

P-Creatinine > 62 µmol/l:

eGFR = 144 x (P-Creatinine /(0.7 x 88.4))-1.209 x 0.993^age

Male

P-Creatinine ≤80 µmol/l:

eGFR = 141 x (P-Creatinine /(0.9 x 88.4))-0.411 x 0.993^age

P-Creatinine > 80 µmol/l:

eGFR = 141 x (P-Creatinine /(0.9 x 88.4))-1.209 x 0.993^age

3. The manuscript does not clarify whether patients had chronic kidney disease, defined as kidney damage or decreased kidney function persisting for three or more months. A single instance of slightly elevated creatinine does not accurately reflect true renal function.

Author Response: We thank the reviewer for bringing this lack of clarity to our attention. We have added a description of sample availability to the methods section [lines 106-112].

“In this study, we define a 30-day preoperative eGFR as the mean of all eGFR measurements conducted in the month leading up to the operation. The formulas used by the regional hospitals to calculate eGFR is available in S1 File. The median number of eGFR measurements per patient in the month up to operation was 2 (IQR 1-3). The 30-day preoperative eGFR was used to define whether a patient had low renal function or normal renal function. In accordance with Kidney Disease: Improving Global Outcomes (KDGIO) guidelines (https://kdigo.org) patients were categorized as having low renal function if their average eGFR was below 60 mL/min/1.73m2.

We acknowledge the reviewer’s concern in relation to assessing renal function based on a single eGRF measurement. The majority of patients had two or more eGFR measurements made in the 30-day timeframe before operation (n = 300, 62.4%). While we agree that the 30-day eGFR assessments based on a single measurement are less robust, we still believe that the affected patients contribute with important information. Furthermore, as the renal and fluid status measurements conducted in the run-up to operation were made at the treating clinician’s discretion, excluding patients with a single measurement would exacerbate the problem of confounding by indication. Due to these reasons, we abstain from excluding the patients.

In light of the points raised by the reviewer, we have added a section in the discussion to highlight this variance in sample numbers more clearly [line 223-229].

“Our study focused on laboratory test results in the short 30-day period before curative intended surgery as this period is the standard length of the Danish preoperative CRC cancer work-up program. However, this has the limitation of not meeting KDGIOs recommended 3-month sampling period for assessing long-term renal function. While most of our patients had multiple eGFR measurements in the 30-day period, a minor fraction had just a single eGFR measurement (n = 181 37.6%). These singular measurements provide important information, yet will not account for the patient’s biological variance in eGFR.”

4. Figure 1 indicates that only a small number of patients had creatinine levels above the normal reference range or an eGFR below the normal reference range. Drawing broad conclusions from such limited data may not be justified. The same concern applies to liver function tests, particularly serum bilirubin levels.

Author Response: We agree with the reviewer that our study offers limited information on patients with extremely low renal and liver function. However, it is highly relevant for the cfDNA/ctDNA community to get a better understanding of the relationship between the natural variation in renal function of CRC patients eligible for surgery and ctDNA test results. The cohort of this study is an unselected representative group of stage I-III CRC patients who are undergoing curative intended surgery. It would be possible to conduct a similar study on patients with end-stage renal and liver failure, but it would offer no actionable information for cancer patients undergoing curative intended treatments, as patients with extremely low renal and liver function are not candidates for surgery. We now address this important topic in the discussion section. [lines 202-204 and lines 236-237]

“Patients with much lower renal and liver function, than those included in this study, can be found in Denmark, but these would very rarely be candidates for curative intended surgery in the Danish healthcare system.”

“For now, it is encouraging that abnormal liver and renal function is of no great concern when conducting ctDNA testing, as the ctDNA clearance can be considered robust across a wide range of test results in CRC patients undergoing curative intended treatment.”

5. It would be beneficial to categorize patients with eGFR below 60 into subgroups based on the severity of renal impairment (e.g., eGFR 45-59, 30-44, etc.) to determine whether ctDNA levels are affected at specific eGFR threshold.

Author Response: We appreciate the reviewer’s warranted interest in this topic. We agree that a subgroup analysis of eGFR measurements is interesting. We originally chose not include these analyses in the manuscript, because there are very few patients in the severely impacted categories. However, based on the reviewer’s feedback we have now included the analyses in the supplementary materials (S1 and S2 Figures).

In summary, this manuscript does not offer any clinically valuable information.

Author Response: We respectfully disagree with this assertion. The field of ctDNA testing is currently undergoing rapid development. Uncovering possible sources off natural variation in ctDNA and cfDNA measurements remains an important concern for the field. Especially as attempts are made at potentiating the ctDNA levels in the bloodstream for sampling purposes.

Reviewer #2: The manuscript reports an interesting concept of impact of variation in renal and liver functions on the detection of ctDNA/cfDNA in patients with resectable colorectal cancer. The conclusion is that there is no such effect. I have the following comments.

1. The only major limitation in this study is that the majority of the participants have their renal/liver functions within reference range and hardly any participant has severe renal or liver dysfunction. This should be clearly highlighted as a limitation and reason for contradiction from the the pre-clinical and previously published reports.

Author response: We are thankful for the reviewer’s insightful and relevant suggestion. We do believe that our cohort is well suited to investigate whether ctDNA and cfDNA clearance inhibition occurs naturally in stage I-III CRC patients undergoing curative intended surgery. We agree wholeheartedly that it is important to communicate that our study does not provide much information on whether severe renal and liver dysfunction could inhibit cfDNA and ctDNA clearance. We have amended the discussion section to more clearly address the low prevalence of severe renal and liver function in our cohort. [lines 202-204]

“Patients with much lower renal and liver function, than those included in this study, can be found in Denmark, but these would very rarely be candidates for curative intended surgery in the Danish healthcare system.”

2. It appears the bloods samples for ctDNA were taken both pre and post-surgery, but only pre-op data is presented. Did they compare post op ctDNA with post-op renal/liver functions?

Author response: We appreciate the reviewer’s interest in preoperative ctDNA and postoperative renal/liver functions. Our study elects to focus on preoperative laboratory biomarkers and ctDNA tests. Preoperatively, patients have their liver and renal function systematically assessed as part of the clinical workup before surgery. In the postoperative situation, liver and renal function are much less systematic, meaning that indication, intervention, number of measurements and the timing vary considerably. A large fraction of these measurements was made after clinical intervention and outlier values were often quickly normalized. Accordingly, registry data of post-operative clinical renal and liver assessments are not an appropriate data source for making generalizable correlations between cfDNA/ctDNA and renal/liver function. In our view, elucidating the immediate post-operative ctDNA and cfDNA metabolism of cancer patients would require a clinical study specifically designed to prospectively and systematically gather the needed data (liver/renal/cfDNA/ctDNA measurements made at the same time-point), which is beyond the scope of this article.

The reviewer’s comment made us reflect on the methods section describing the blood samples available for ctDNA analysis. [Line 79 in original manuscript]

“As part of the study, patients had blood collected prior to and after surgery, with the aim to retrospectively measure cfDNA and ctDNA.”

As our study does not involve the postoperative samples, we have elected to remove the mentioning of these samples to avoid confusion. [Line 74]

Editorial office revision requests

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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Author response: We thank the editor for the resources provided and have used them to revise the manuscript to follow PLOS ONE’s style requirements: A list of changes is given below:

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2. Please update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex.

Author response: We have elected to resubmit the manuscript in DOCX format. The original PDF submission was a mistake due to misunderstanding the author guidelines. We apologize for the mistake and are grateful for the opportunity to correct it.

3. In the online submission form you indicate that your data is not available for proprietary reasons and have provided a contact point for accessing this data. Please note that your current contact point is a co-author on this manuscript. According to our Data Policy, the contact point must not be an author on the manuscript and must be an institutional contact, ideally not an individual. Please revise your data statement to a non-author institutional point of contact, such as a data access or ethics committee, and send this to us via return email. Please also include contact information for the third party organization, and please include the full citation of where the data can be found.

Author response: We have edited the manuscript so that the contact point is The Department of Molecular Medicine at moma@rm.dk and included a description of how to use the mail to request data access.

“Because the data contains sensitive personal patient information, it cannot be made publicly available. Access to data requires that the data requestor (legal entity) enter into Collaboration and Data Processing Agreements, with the Central Denmark Region (the legal entity controlling and responsible for the data). Inquiries for access can be addressed to the Data Access Committee at Department of Molecular Medicine, Aarhus University Hospital (contact via moma@rm.dk).”

Attachment

Submitted filename: Response to Reviewers.docx

pone.0319194.s010.docx (28.8KB, docx)

Decision Letter 1

Elingarami Sauli

3 Dec 2024

PONE-D-24-32619R1The role of renal and liver function in clinical ctDNA testingPLOS ONE

Dear Dr. Andersen,

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.

==============================

The authors should clearly compare post op ctDNA with post-op renal/liver functions, to show their difference with pre op data.

==============================

Please submit your revised manuscript by Jan 17 2025 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,

Elingarami Sauli, PhD

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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: No

Reviewer #2: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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: This manuscript does not contribute any significant useful information to the literature as most patients had normal liver and renal function. Besides the timepoint of ctDNA/cfDNA measurement is clinically irrelevant.

Reviewer #2: The authors have addressed the concerns adequately. Though the impact of the findings are unlikely to be significant to current practice, the manuscript on its own is technically well written otherwise. I have no other issues with the manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PLoS One. 2025 Feb 25;20(2):e0319194. doi: 10.1371/journal.pone.0319194.r005

Author response to Decision Letter 1

17 Jan 2025

Editorial office revision requests

1. The authors should clearly compare post op ctDNA with post-op renal/liver functions, to show their difference with pre op data.

Author response: We concur that there is justified interest in exploring the correlation between renal/liver functions and cfDNA/ctDNA also in the postoperative setting. However, we believe there are serious limitations and challenges to using the data of this study for such a purpose.

Challenges in postOP analysis of the correlation between ctDNA and renal/liver functions:

As colorectal cancer (CRC) patients are often cured by surgery, most colorectal cancer patients have no ctDNA in the blood after surgery. Additionally, our cohort consists mainly of low-risk patients (pT1-3pN0, 63.6%). Of the 846 person cohort, only 62 (7.24%) have a positive ctDNA call in the postoperative sample. Furthermore, for the few patients who have residual disease after surgery, the initiation of adjuvant chemotherapy (ACT) impacts the residual tumor burden, leading to a lowering, and potentially elimination, of the ctDNA fraction in blood. Therefore, only blood samples collected prior to initiation of ACT are relevant for analysis of the correlation between ctDNA and renal/liver function.

Altogether, the number of patients for which we have suited samples with ctDNA detected before ACT initiation is very low (n = 59). This is too few for statistical analysis.

Challenges in postOP analysis of the correlation between cfDNA and renal/liver functions:

Postoperative cfDNA analyses are complicated by the temporary surgical trauma-induced surge in normal DNA circulating in the blood [1]. We have previously reported that the normal DNA level in the blood increases a median of 3.6-fold shortly after the surgical trauma. After this, the concentration slowly returns to normal level, which is typically reached around week four after surgery. Therefore, the correlation between cfDNA level and renal/liver function can only be studied in samples collected after week 4.

Also for cfDNA analysis, initiation of ACT is challenge. The ACT likely impacts the hematopoietic cellular turnover, and thereby the normal DNA level in blood. ACT is also likely to impact the renal/liver cfDNA clearance mechanisms. Therefore, it is futile to investigate the correlation between cfDNA and renal/liver functions after ACT initiation. In Denmark, the recommendation is to start ACT between week 4 and week 6, and the median ACT start date in our cohort is 31 days after surgery. Consequently, the window is very narrow for collection of samples that can be meaningfully used to assess the correlation between cfDNA and renal/liver function.

Challenges with PostOP analysis of laboratory biomarkers of renal/liver function:

There are also challenges related to the laboratory biomarkers in the postoperative setting. In the preoperative setting, patients have laboratory tests performed to map the baseline renal and liver function prior to deciding the treatment plan. The situation is very different in the immediate postoperative setting. There, renal/liver function is measured because the patient exhibits symptoms that compels a clinician investigate for treatment indication. Therefore, there is significant “confounding by indication” in the available data. Furthermore, as the laboratory test results are made by indication they are monitored closely and clinical intervention is immediately initiated for patients with deviant values. Effectively, the analysis by indication and the subsequent interventions make the laboratory test-results useless for analyses of any natural correlation between cfDNA and laboratory biomarkers of renal/liver function.

To illustrate the challenge, we have included a figure with serial potassium values from 6 patients measured over a period from operation to day 60. The oscillating potassium values are consistent with clinical intervention after deviant values were registered. Consequently, for a meaningful analysis of the correlation between cfDNA and renal/liver function, the cfDNA and renal/liver laboratory biomarker needs to be measured very closely in time.

Figure: line plots showing potassium levels for six patients in the cohort. Each line is a single patient’s potassium levels from day of surgery until 60 days after. The red dotted lines signify upper and lower reference range bounds.

We explored how many of our patients had blood samples available that fitted the above requirements (collected after week 4 and prior to initiation of ACT) and for which renal/liver function laboratory test results were available on same day or up to 5 days after. The upper time limit for cfDNA sample inclusion was 57 days after surgery, which is a 30 day time period from the end of week 4.

In total, we had 253 cfDNA samples four weeks after surgery and before initiation of ACT. Of these, 33 (13%) patients had laboratory markers taken in the valid timeframe, which is too few for sound statistical analysis. Properly addressing how postoperative renal and liver function impacts ctDNA and cfDNA metabolism will require a new prospective clinical trial, designed specifically for this purpose. Such a trial is beyond the scope of this article.

While we maintain that our dataset is suboptimal to address the question. We acknowledge that this question is of interest to the reviewer and the readership of PlosOne. Therefore, we have incorporated an analysis of postoperative cfDNA clearance in the supporting information section. This includes methodology, figures, full regression results and a discussion of the limitations and challenges involved in investigating the postoperative metabolism of cfDNA and ctDNA. We refer to S2 file in the revised manuscript [lines 195-197].

“In addition we conducted a supplementary investigation of cfDNA levels in the postoperative setting (S2 File) and found no significant association between postoperative cfDNA level and biomarkers of renal and liver function”

We hope that presenting this analysis will be of use to researchers wishing to investigate this subject in more detail in the future.

1. Henriksen TV, Reinert T, Christensen E, Sethi H, Birkenkamp-Demtröder K, Gögenur M, et al. The effect of surgical trauma on circulating free DNA levels in cancer patients—implications for studies of circulating tumor DNA. Molecular Oncology. 2020;14(8):1670-9.

Attachment

Submitted filename: Response_to_reviewers_auresp_2.docx

pone.0319194.s011.docx (28.8KB, docx)

Decision Letter 2

Elingarami Sauli

29 Jan 2025

The role of renal and liver function in clinical ctDNA testing

PONE-D-24-32619R2

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Acceptance letter

Elingarami Sauli

PONE-D-24-32619R2

PLOS ONE

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Association of eGFR with cfDNA level stratified by age.

    Figures showing grouped eGFR levels and continuous cfDNA levels stratified for patient age. Associations were calculated with a paired Wilcoxon signed rank test.

    (EPS)

    pone.0319194.s001.eps (874.5KB, eps)
    S2 Fig. Association of eGFR with ctDNA level stratified by age.

    Figures showing grouped eGFR levels and continuous ctDNA levels stratified for patient age. Associations were calculated with a paired Wilcoxon signed rank test.

    (EPS)

    pone.0319194.s002.eps (821.9KB, eps)
    S1 Text

    S1 Table. Reference ranges for the laboratory tests. S2 Table. Number of patients for whom a specific laboratory test was available. S3 Table. Full log-log linear regression results for cfDNA. S4 Table. Full log-log linear regression results for ctDNA. S5 Table. Full binomial logistic regression results for ctDNA detection compared to laboratory test result. S6 Table. Full binomial logistic regression results for ctDNA detection compared to reference range category of laboratory test. S7 Table. Full binomial logistic regression results for ctDNA and cfDNA compared to low and high renal function.

    (XLSX)

    pone.0319194.s003.xlsx (42.5KB, xlsx)
    S1 File. Description of eGFR calculation formulas.

    (DOCX)

    pone.0319194.s004.docx (19.6KB, docx)
    S2 File. Postoperative analysis of cfDNA and laboratory biomarkers of renal and liver function.

    (DOCX)

    pone.0319194.s005.docx (41.9KB, docx)
    S3 Fig. Potassium_chart.

    (EPS)

    pone.0319194.s006.eps (220.7KB, eps)
    S4 Fig. Postop_cohort_funnel.

    (EPS)

    pone.0319194.s007.eps (135.1KB, eps)
    S5 Fig. Postop_cfDNA_linear_regression.

    (EPS)

    pone.0319194.s008.eps (489.9KB, eps)
    S6 Fig. Postop_renal_function.

    (EPS)

    pone.0319194.s009.eps (80.1KB, eps)
    Attachment

    Submitted filename: Response to Reviewers.docx

    pone.0319194.s010.docx (28.8KB, docx)
    Attachment

    Submitted filename: Response_to_reviewers_auresp_2.docx

    pone.0319194.s011.docx (28.8KB, docx)

    Data Availability Statement

    Because the data contains sensitive personal patient information, it cannot be made publicly available. Access to data requires that the data requestor (legal entity) enter into Collaboration and Data Processing Agreements, with the Central Denmark Region (the legal entity controlling and responsible for the data). Inquiries for access can be addressed to the Data Access Committee at Department of Molecular Medicine, Aarhus University Hospital (contact via moma@rm.dk).

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