Assessing the Presence of CT-DNA in Lymphoma Associated HLH

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A variety of constitutive and acquired risks trigger HLH. In general, infants have inherited T- and NK-cell defects impairing cytotoxic function, while adults are most likely to suffer from lymphoma, most commonly diffuse large B cell lymphoma (DLBCL), Hodgkin lymphoma and T-cell lymphoma. Patients with underlying lymphoma have the worst survival rates (West et al J Intern Med 2021). Diagnosis of lymphoma is challenging due to severe sepsis-like presentation, meaning CT-PET and early biopsy may not be possible. Patients with delayed/no diagnosis frequently receive empirical chemotherapy, delaying diagnosis, inadequately treating underlying lymphoma and potentially worsening infections. Epidemiological studies have failed to show improvements in survival in patients with HLH, whilst also confirming a significant increase in the number of diagnoses (West et al J Intern Med 2021). Improvements in rapidly diagnosing cases of lymphoma driving HLH would result in better outcomes due more rapid (immuno-)chemotherapy administration. Circulating tumour DNA (ctDNA) are DNA isolated from blood, originating from the apoptosis/necrosis of cancerous cells. ctDNA reflects the entire tumour genome and is referred to as a "liquid biopsy". These techniques are under investigation in several lymphomas, and DLBCL-specific mutations can be detected and quantified using ctDNA, with studies using quantification as a strategy to monitor response to therapy (Kurtz et al J Clin Onc 2018). Similarly, ctDNA mutations can be identified in Hodgkin lymphoma (Spina et al Blood 2018) and T-cell lymphoma (Ottolini et al Blood advances 2020). Capitalising on the success of the DLBCL Interim Response Evaluation for Customised Therapy (DIRECT) study, existing infrastructure in Cambridge will be used to conduct a feasibility study assessing whether ctDNA from patients with HLH with underlying lymphoma is viable in contributing to diagnoses. Blood samples from patients with HLH will have ctDNA and granulocytes extracted and stored. Once lymphoma is confirmed, the biopsy will be requested and ctDNA, granulocytes and biopsy from each patient will be interrogated using shallow whole genome sequencing (WGS; 0.1x) and a high-sensitivity, targeted sequencing panel termed LyVE-Seq (\~2000x). This panel includes coding regions of 122 genes implicated as drivers of DLBCL, in addition to translocation hotspots for BCL6/MYC. For non-DLBCL, the existing panel will be modified to include 150 genes recurrently mutated across all lymphoma subtypes, ordered as a focused panel from Twist Bioscience. Demographic/laboratory data will be requested and will be integrated with information from clinical risk scores, tumour genotype, and radiology (CT/PET-CT). These invaluable clinical samples will be stored and may be used for future research as, to date, there is no data for ctDNA in the context of malignancy associated HLH and the study is highly exploratory.