Liquid biopsy in lymphoma: Molecular methods and clinical applications

Abstract

In this article, we broadly review the application of cfDNA analysis to the diagnosis and management of lymphoma. We introduce the advantages of cfDNA measurement over conventional tissue biopsy and describe how cfDNA may be utilized for both genotyping and detection of minimal residual disease. First, we discuss genotyping, beginning with differences in identifying mutations from the blood plasma vs. from circulating cells. We review the technical distinctions between PCR- and NGS-based assays and describe two important applications of NGS-based cfDNA tests, namely the identification of resistance mutations and classification of disease subtype. We discuss difficulties in genotyping diseases with low burden of tumor cells and the application of cfDNA assays in these contexts. Second, we describe the utility of ctDNA measurement in assessing MRD. We cover recent advances in the assessment of pre-treatment disease burden as a prognostic biomarker, detection of molecular response to therapy, and early detection of relapsing disease. Third, we explore select emerging areas of research in ctDNA technologies that show promise in boosting the performance of existing ctDNA-based assays. These include cell-free DNA fragment structure analysis or 'fragmentomics', epigenetic modifications, and novel circulating analytes such as tumor-educated platelets and extracellular vesicular DNA. We also discuss alternative analytes to blood plasma for tumor detection, such as urine, saliva, and stool. Finally, we present a case that highlights potential applications of ctDNA approaches to the management of patients with lymphoma, while also defining important prerequisite advances before this can be fully realized. We close with a look to the future of cfDNA applications, outlining one potential timeline and path forward towards routine clinical application.

Keywords: Cell-free DNA; Circulating tumor DNA; Liquid biopsy; Lymphoma; Minimal residual disease; Precision medicine.

Figure 1.. Circulating Tumor DNA Applications in Lymphoma

This figure shows a timeline of select past, present, and expected key milestones in liquid biopsies as applied to lymphoma. We show seminal studies marking currently achieved milestones, as well as attempt to identify one potential roadmap to the routine application of ctDNA in clinical practice.

Figure 2:. Tumor heterogeneity and response to treatment

A; Pre-treatment PET/CT scan in newly diagnosed lymphoma demonstrates advanced stage disease. B; Inguinal lymph node biopsy demonstrating low-grade follicular NHL, H&E stain (x40 magnification). C; Inguinal lymph node biopsy demonstrating low-grade follicular NHL, BCL-2 IHC stain (x60 magnification). D; Gastric ulcer biopsy demonstrating high grade B-cell lymphoma DLBCL, H&E stain (x60 magnification). E; End of treatment PET/CT scan demonstrates near complete remission. F; MRI scan after NHL treatment demonstrates new peri-ventricular white matter lesions suggestive of NHL relapse.