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Trial registered on ANZCTR


Registration number
ACTRN12622001326785
Ethics application status
Approved
Date submitted
20/09/2022
Date registered
12/10/2022
Date last updated
12/10/2022
Date data sharing statement initially provided
12/10/2022
Type of registration
Prospectively registered

Titles & IDs
Public title
Clinical validation of circulating cell free DNA [ccfDNA] as a biomarker of metabolic health
Scientific title
Clinical validation of circulating cell free DNA [ccfDNA] as a biomarker of metabolic health
Secondary ID [1] 307996 0
NIL KNown
Universal Trial Number (UTN)
Trial acronym
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Metabolic Health 327667 0
Obesity 327873 0
Condition category
Condition code
Metabolic and Endocrine 324751 324751 0 0
Metabolic disorders
Diet and Nutrition 324954 324954 0 0
Obesity

Intervention/exposure
Study type
Observational
Patient registry
False
Target follow-up duration
Target follow-up type
Description of intervention(s) / exposure
This trial comprises a dual site, non-intervention cross-sectional observational study. The study population will comprise of up to 100 obese individuals of unknown metabolic health. The primary objective is to validate a cell free DNA based blood assay for its ability to identify individuals of poor metabolic health. Participants will undergo a single face-to-face assessment at CSIRO's Adelaide (SAHMRI) and Westmead trial facilities of approximately 1 hour duration with no further visits required. Before attending, subjects will complete a screening questionnaire to ensure suitability, which will be verified by the clinic team on arrival. Screening and anthropomorphic measurements will be performed by the clinical trial coordinator, all blood collections will be made by a trained Registered Nurse and/or phlebotomist. Participants will be classified as metabolically healthy or metabolically abnormal obese. Groups will be further assessed for indicators of liver health (fibrosis and fatty liver). Health status will be assessed using the below set of biomarkers:
o Blood Pressure (mmHg)
o Blood Glucose concentration
o Blood HbA1c concentration
o Blood Platelet Count
o Serum Triglycerides
o Serum HDL-Cholesterol
o Serum LDL-Cholesterol
o Serum total Cholesterol
o Serum Insulin concentration
o Serum hsCRP concentration
o Serum AST concentration
o Serum ALT concentration
o Serum GGT concentration
o Serum albumin concentration
o Waist circumference (cm)
o Hip circumference (cm)
o BMI (kg/m2), Weight (kg), Height (cm)

A further blood sample will be provided for the purpose of epigenetic analysis of the cell free DNA. Cell free DNA concentrations will be compared to metabolic and liver health biomarkers above to assess the clinical validity of a novel blood test for metabolic health.
Intervention code [1] 324452 0
Diagnosis / Prognosis
Comparator / control treatment
Participants to be separated into two groups based on metabolic biomarkers (Metabolically Healthy Obese and Metabolically Abnormal Obese). Comparison in cell free DNA concentrations originating from the Liver and the Subcutaneous Adipose tissues will be compared between groups.
Control group
Active

Outcomes
Primary outcome [1] 332571 0
Sensitivity of ccfDNA concentration as measured by our assay to detect metabolically abnormal obesity among a blind testing population comprised of both metabolically healthy and abnormal obese participants. The true positive rate for metabolic abnormality will be assessed for the cell free DNA concentration levels from blood samples using a novel droplet digital PCR assay, compared to the published standards (criteria for metabolically healthy obese individuals as defined in the literature [Aguilar-Salinas et al 2008, ATP III 2001, Karelis et al 2004, Lynch et al 2009, Meigs et al 2006, Wildman et al 2008 and Zembic et al 2021])."
Timepoint [1] 332571 0
This is a single time point study with only one clinical visit in which all metabolic and liver profiling will be performed.
Primary outcome [2] 332758 0
Specificity in detecting metabolically abnormal obese individuals amongst the blind testing population comprised of participants who are either metabolically healthy obese and those who are metabolically abnormal. The true negative rate for metabolic abnormality will be assessed for the cell free DNA concentration levels from blood samples using a novel droplet digital PCR assay, compared to the published standards (criteria for metabolically healthy obese individuals as defined in the literature [Aguilar-Salinas et al 2008, ATP III 2001, Karelis et al 2004, Lynch et al 2009, Meigs et al 2006, Wildman et al 2008 and Zembic et al 2021]).
Timepoint [2] 332758 0
This is a single time point study with only one clinical visit in which all metabolic and liver profiling will be performed.
Secondary outcome [1] 413955 0
The secondary outcome will be to identify any correlation between the concentration of cell free DNA (using the above described ccfDNA ddPCR assay) and estimated levels of liver fibrosis. This outcome will be used to establish the role of this assay in future studies for liver health. Liver fibrosis levels will be estimated using the below set of scores using the following inputs:

NAFLD Fibrosis Score (NFS) inputs:
Age in years, BMI, diabetes diagnosis, Blood Aspartate Aminotransferase (AST) concentration, Blood Alanine Aminotransferase (ALT) concentration, platelet count, serum albumin concentration

FIBrosis-4 (FIB-4) inputs:
Age in years, AST concentration, ALT concentration, platelet count

AST to Platelet Ratio Index (APRI) inputs:
AST concentration, platelet count

The concentration of cell free DNA will be used to establish a relationship between liver health and cfDNA released into the bloodstream to support further studies.
Timepoint [1] 413955 0
This is a single time point study with only one clinical visit in which all metabolic and liver profiling will be performed.
Secondary outcome [2] 414644 0
A further secondary outcome will be to identify any correlation between the concentration of cell free DNA (using the above described ccfDNA ddPCR assay) and estimated severity of fatty liver. This outcome will be used to establish the role of this assay in future studies for liver health. Fatty liver severity will be estimated using the Fatty Liver Index (FLI) using the following inputs:

Blood triglyceride concentrations
BMI
Blood Gamma Glutamyl Transferase (GGT) concentration
Waist circumference (cm)

The concentration of cell free DNA will be used to establish a relationship between liver health and cfDNA released into the bloodstream to support further studies.
Timepoint [2] 414644 0
This is a single time point study with only one clinical visit in which all metabolic and liver profiling will be performed.

Eligibility
Key inclusion criteria
1. Willing to provide written Informed Consent
2. Aged from >=18 to <56 years of age at clinic visit
3. BMI >=30 and <=35 kg/m2 confirmed at visit
4. Willing to fast at least 10 hours before attending clinic visit
Minimum age
18 Years
Maximum age
55 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
1. Currently pregnant and/or nursing (lactating)
2. Diagnosis of insulin resistance, diabetes (Type I, Type II and/or Gestational Diabetes), cancer including skin cancer, and immunological disease
3. Current or prior chemotherapy/radiotherapy treatment
4. Current use or use of the following medications in the last 84 days prior to clinic visit:
• Metformin
• Fortamet
• Glucophage
• Glucophage XR
• Glumetza
• Riomet
• N,N-dimethylbiguanide
5. Experienced physical injury 14 days prior to clinic visit that meets any of the following:
• Required medical assessment and/or treatment
• Resulted in pain, bruising and/or sensitivity lasting longer than 14 days or is currently being experienced.
• Resulted in limited movement and mobility
6. Average Systolic BP >=180mmHg or Diastolic BP >= 110mmHg
7. In the opinion of the Principal Investigator, unable to comply and/or experience distress with study procedures.

Study design
Purpose
Screening
Duration
Cross-sectional
Selection
Defined population
Timing
Prospective
Statistical methods / analysis
Sample Size Calculation
Analysis of our droplet digital PCR (ddPCR) assay in an existing cohort demonstrated an effect size (Cohen’s d) of 0.7 between the Metabolically Healthy Obese (MHO) and Metabolically Abnormal Obese (MAO) groups. To achieve a study with 80% power and an alpha of 0.05 the effect size of our assay suggests we require no less than 31 participants in each of the MHO and MAO groups. Incidence of MHO varies significantly across the published definitions and between studies, occurring with much lower incidence when compared to MAO. Exploring datasets from previous clinical trials in our Research Clinic indicated that MHO individuals made up between 12-30% of the obese population and that relying on a random collection strategy would likely require at least 200 participants which is not feasible (cost and time). Within a restricted cohort defined as having a BMI between 30-35 and aged < 55 years the prevalence of MHO is much higher, between 18-50%. With this restriction a much smaller cohort may be collected to achieve statistical power. Therefore, using these BMI and age criteria, to guarantee a population of at least 31 in each group we estimated that we need to recruit >86 participants with 100 budgeted.

Analysis of liver health outcomes in this study is a discovery aim. This work will generate new data that will be used to establish effect size and statistical associations between liver health scores and the output of the ccfDNA ddPCR assay. As such, statistical power calculations cannot be performed for liver outcomes.

Statistical Analysis Plan
Statistical analysis will be performed on the de-identified dataset by the CSIRO Sydney Team. Sydney team members will be blinded to Metabolic health status (as defined by the comprehensive metabolic profiling data). To achieve blinding, metabolic profiling data will be withheld from the Sydney Statistical team during the initial ddPCR analysis and only released after the ccfDNA experiments have been completed (i.e., after MHO/MAO ccfDNA predictions have been made). Patients will be classified using a simple threshold count value from the ddPCR data. The biomarker targets in the ddPCR assay will be analysed individually and as a mean to establish the relationship between ccfDNA and metabolic health status. Additionally, a logistic regression model will be trained on the existing EpiSCOPE cohort data and applied to this study. Accuracy of model calls will be assessed against metabolic health status after unblinding. After unblinding further exploratory analysis will be performed to identify correlations between ddPCR, metabolic health status and other clinical factors reported in the participants medical history (e.g., use of prescribed drugs such as statins) This analysis will be performed using common statistical programming languages including R and python.
Assessment of liver outcomes is an exploratory component of this study with the aim to discover new underlying associations between our biomarkers and progression of metabolic disease(s). Following classification of the cohort by ddPCR analysis and MHO/MAO assessment, NFS, FIB-4, FLI and APRI indexes will be calculated and the presence of fatty liver and fibrosis determined using published cut-offs. Liver and Visceral Adipose ddPCR count data will be correlated with scores individually and as a mean. Further, ‘positive’ and ‘negative’ populations (using ddPCR analysis above) will be assessed for enrichment of fibrosis/fatty liver in each group Fisher Exact tests will be used for pairwise comparisons between groups.

Recruitment
Recruitment status
Not yet recruiting
Date of first participant enrolment
Anticipated
31/10/2022
Actual
Date of last participant enrolment
Anticipated
31/10/2023
Actual
Date of last data collection
Anticipated
31/10/2023
Actual
Sample size
Target
100
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
NSW,SA

Funding & Sponsors
Funding source category [1] 312262 0
Government body
Name [1] 312262 0
Commonwealth Scientific and Industrial Research Organisation
Country [1] 312262 0
Australia
Primary sponsor type
Government body
Name
Commonwealth Scientific and Industrial Research Organisation
Address
Building 101
2 - 40 Clunies Ross Street
Acton ACT 2601
Australia
Country
Australia
Secondary sponsor category [1] 313802 0
None
Name [1] 313802 0
Address [1] 313802 0
Country [1] 313802 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 311635 0
CSIRO Health and Medical Human Research Ethics Committee
Ethics committee address [1] 311635 0
Ethics committee country [1] 311635 0
Australia
Date submitted for ethics approval [1] 311635 0
25/01/2022
Approval date [1] 311635 0
21/07/2022
Ethics approval number [1] 311635 0
2022_006_HREC

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 121798 0
Dr Warwick Locke
Address 121798 0
CSIRO Molecular Diagnostics
Level 3, Innovation Quarter
160 Hawksbury Road
Westmead, NSW, 2145
Country 121798 0
Australia
Phone 121798 0
+61 294908676
Fax 121798 0
Email 121798 0
warwick.locke@csiro.au
Contact person for public queries
Name 121799 0
Warwick Locke
Address 121799 0
CSIRO Molecular Diagnostics
Level 3, Innovation Quarter
160 Hawksbury Road
Westmead, NSW, 2145
Country 121799 0
Australia
Phone 121799 0
+61 294908676
Fax 121799 0
Email 121799 0
warwick.locke@csiro.au
Contact person for scientific queries
Name 121800 0
Warwick Locke
Address 121800 0
CSIRO Molecular Diagnostics
Level 3, Innovation Quarter
160 Hawksbury Road
Westmead, NSW, 2145
Country 121800 0
Australia
Phone 121800 0
+61 294908676
Fax 121800 0
Email 121800 0
warwick.locke@csiro.au

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
Yes
What data in particular will be shared?
Individual participant data required to support research claims and/or intellectual property claims may be published in a de-identified, fully anonymised format as part of a peer reviewed research article and/or patent. Items shared will be limited to that which is required to support the claims made.
When will data be available (start and end dates)?
De-identified data would be available indefinitely immediately upon publication of any scientific findings in a high impact, peer reviewed journal.
Available to whom?
Data would be available to anyone who wishes to access it as part of a scientific journal article (via the publisher) or patent.
Available for what types of analyses?
Data will be available for free use. Data is shared primarily to demonstrate the validity of our claims and findings or to support replication by independent researchers.
How or where can data be obtained?
Upon study completion it is anticipated that, intellectual property considerations notwithstanding, a de-identified dataset will be stored on CSIRO’s Data Access Portal (DAP) at https://data.csiro.au/ to enable future discovery and sharing of de-identified data. Data will also be shared as supplementary materials in a high-impact, peer-reviewed scientific journal.


What supporting documents are/will be available?

Doc. No.TypeCitationLinkEmailOther DetailsAttachment
17147Study protocol  warwick.locke@csiro.au
17148Informed consent form  warwick.locke@csiro.au
17155Ethical approval  warwick.locke@csiro.au



Results publications and other study-related documents

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