2025 Arthritis Australia Project Grant Scientific Report

Background

Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting ~1% of Australians, wherein immune cells attack the synovial tissue (joint lining). In RA, the synovial tissue becomes heavily infiltrated with diverse inflammatory cells which destroy the underlying cartilage and bone. This leads to swelling, pain, and irreversible joint deformities, severely reducing quality of life1. While the presentation and trajectory of RA is incredibly heterogeneous, disease course is generally beset by repeated flares, with each flare followed by progressive joint damage and reduced functional ability2. Long-term complications of RA include early mortality, a pronounced (~50%) increase in cardiovascular disease (CVD)3 risk, and increased risk of haematologic complications including lymphoma and myeloid neoplasms (a group of hematologic cancers).

Our previous research (published in Blood4) revealed the importance of acquired (somatic) mutations in RA. These mutations typically affect the haematopoietic progenitor stem cells of the bone marrow where they confer survival or proliferative advantage, resulting in expansion of mutated progeny (‘clonal haematopoiesis’ (CH)). CH is associated with increased risk of haematological malignancies and inflammation5. Our group previously demonstrated4 6 that CH strikingly stratifies RA subtypes, drives inflammation, and treatment resistance. These findings underpin our paradigm-changing conceptualisation of RA pathogenesis and progression, whereby (a) relentless autoantigen exposure drives local and systemic inflammation and promotes immune cell proliferation, which in turn (b) accelerates CH incidence7 and dysregulates immune cells, resulting in a feedback loop that exacerbates disease8 9 and compounds CVD risk in RA.

In this project, we sought to specifically investigate the relationship between CH and cardiovascular outcomes in our cohort of patients enrolled in the “Methotrexate, blood pressure, and arterial function in RA” study (trial number NCT03254589).

Project Aims:

Our project was built around three main aims:

1. To assess CH prevalence and type in the NCT03254589 RA cohort, and in early vs. established RA.
2. To relate CH status with clinical outcomes, including disease activity, HAQ, response to treatment, markers of inflammation, prevalence of comorbidities and robust markers of atherosclerosis and CVD risk (i.e., blood pressure, augmentation index and pulse wave velocity).
3. To demonstrate the functional consequences of CH in the myeloid compartment of RA patients.

Summary of completed work

Aim 1.

For this aim, we sought and attained local ethics committee approval to access and work with the samples from the NCT03254589 RA cohort. Following this, we isolated genomic DNA using the Qiagen DNeasy Blood and Tissue extraction kit. DNA quality and quantity was 2

measured via NanoDrop, and samples consisting of 1 μg total DNA were sent to SAHMRI for further quality assessment and targeted capture sequencing. Due to delays in the project (outlined below), sequencing took place in January 2026, leading to delays in data processing and downstream analysis, which is ongoing at the time of this report.

Aim 2.

Due to the delays in data analysis from the samples from the NCT03254589 RA cohort, we are yet to conduct comprehensive alignment of patient mutation status with clinical outcomes. However, during the project duration, we were able to establish our clinical data analysis pipeline and relate identified CH-associated mutations detected across two smaller cohorts from the ARBITRATE RA inception cohort housed at Flinders Medical Centre (n=112) and the Royal Adelaide Hospital RA inception cohort, with patient clinical outcomes.

We found somatic mutations at variable allele frequency (VAF) levels ≥0.5% and ≥2% in 27.7% (31/112) and 9.8% (11/112) RA patients, respectively. RA patients over the age of 60 (53.9%) were more likely to have clonal haematopoiesis of indeterminate potential (CHIP) (20.5% vs. 4.1%; P=0.015). This is significantly higher than the reported frequency in an unselected population of 5.6%10. The majority of the 48 identified pathogenic variants involved DNMT3A (47.9%), TET2 (16.7%) and ASXL1 (12.5%). Meanwhile, 9/31 patients (29%) harboured ≥2 mutation variants. While it was not statistically significant (p=0.75), there was a higher prevalence of CHIP in seronegative patients (11.1%) compared to seropositive (8.2%). Patients with high baseline disease activity (DAS28-ESR >5.1) and CHIP or ≥2 distinct CH mutations were less likely to respond to therapy at 6-months (23.5% vs. 7.8%; p=0.1; 23.5% vs. 3.9%; p=0.031, respectively). Strikingly, across this smaller cohort, patients with CHIP had higher rates of incident malignancy (38% vs. 8%; p=0.04), and those with any CH were more likely to have cardiovascular event after RA diagnosis (57% vs. 25%; p=0.05).

Aim 3.

As a supplemental project aim funded locally by the Synovial Tissue Research Group at Flinders University, this aim is currently ongoing.

Changes to the project/Unforeseen circumstances

Throughout the duration of the project, we encountered several notable circumstances that delayed its progress:

• The study had a delayed start due to CIA’s parental leave career interruption (1 month in January 2025)
• The laboratory initially proposed for collaborating with for targeted capture sequencing relocated at the beginning of the project. This led to the decision to switch provider to SA Pathology and delayed optimisation of the new panel in turn led to an extension of the project’s end date from 31 December 2025 to 31 March 2026.

Ongoing work and future directions

The produced raw sequencing data is currently in the final stages of being processed by the genomics team at SA Pathology. We expect to receive the basic processed data in early June, 2026. Work with our bioinformatician will then begin locally, and outputs from this analysis are expected in August 2026. Additional experimentation has also been planned in collaboration with the University of Queensland, where we will undertake whole exome sequencing to further identify the presence of somatic mutations not caught in our targeted capture panel. 3

Significance

There is currently a pressing unmet need to identify the cellular drivers and diversifiers of RA to better explain pathogenesis, enable personalised therapy and improve outcomes. Our preliminary outcomes from this project has provided early evidence implicating somatic mutations in genes related to DNA methylation in contributing to disease severity, treatment resistance, risk of incident malignancy, and cardiovascular in patients with RA. Our identification of these provides the rationale for screening RA patients for target mutations at disease onset and inflection points. Finally, by identifying TET2 mutations in as pathogenic in this cohort, we additionally provide rationale for the application of targeting TET2 mutations in RA to reduce risk of patient co-morbidities and improve patient outcomes.

Outputs from this work

While still an ongoing project, we have produced significant research outputs from the project:

• Early findings were presented at the 2025 American Congress of Rheumatology (ACR) Annual Meeting held in Chicago, USA in October 2025, titled, “Clonal Hematopoiesis of Indeterminate Potential (CHIP) in Rheumatoid Arthritis: associations with treatment response and comorbidities”. This work was selected and highlighted in the ‘Clinical Year in Preview’ session by Professor Tomas Mustelin, a senior rheumatologist from the University of Washington (USA), as an ‘abstract that will shape research in the near future’.
• Early findings were also presented at the Australian Rheumatology Association 2025 Annual Scientific Meeting in Adelaide, SA, Australia, entitled, ‘Clonal Haematopoiesis in Rheumatoid Arthritis: Disease Activity and Comorbidities’. The award for ‘Best Scientific Presentation’ was awarded to this presentation, to Dr. Athena Chin.
• A manuscript of the project findings is currently in preparation.

Consumer Involvement

We received continual consumer input throughout the duration of our project. As a part of the project, we conducted a small-scale consumer survey to guide our research approaches. While this survey is still recruiting, we have received consumer input from 10 patients with RA, providing useful and directly applicable information relevant to our project conduction.

References

1. Rheumatoid Arthritis. Cat. No. 252: Australian Institute of Health and Welfare; 2020 [Available from: https://www.aihw.gov.au/reports/chronic-musculoskeletal-conditions/rheumatoid-arthritis/contents/what-is-rheumatoid-arthritis accessed 15.4.2022 2022.

2. Markusse IM, Dirven L, Gerards AH, et al. Disease flares in rheumatoid arthritis are associated with joint damage progression and disability: 10-year results from the BeSt study. Arthritis Res Ther 2015;17(1):232.

3. England BR, Thiele GM, Anderson DR, et al. Increased cardiovascular risk in rheumatoid arthritis: mechanisms and implications. 2018;361:k1036.

4. Hong LE, Wechalekar MD, Kutyna M, et al. IDH-mutant myeloid neoplasms are associated with seronegative rheumatoid arthritis and innate immune activation. Blood 2024;143(18):1873-77. 4

5. Avagyan S, Zon LI. Clonal hematopoiesis and inflammation – the perpetual cycle. Trends Cell Biol 2023;33(8):695-707.

6. Chin A, Branford S, Small A, et al. Clonal Haematopoiesis of indeterminate potential (CHIP) in Rheumatoid Arthritis. Int Med J 2024

7. Tariq F, Alobaidi B, Xavier M, et al. Clonal Haematopoiesis Associated Somatic Mutations in RA. 2020;79:226-26.

8. Goodnow CC. Multistep pathogenesis of autoimmune disease. Cell 2007;130(1):25-35.

9. Stubbins RJ, Platzbecker U, Karsan A. Inflammation and myeloid malignancy: quenching the flame. Blood 2022;140(10):1067-74.

10. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med 2014;371(26):2488-98.

Acknowledgements

This work was funded by the ARA Trust. We are thankful to Arthritis Australia for their support, as well as all study participants and our consumer advisors.