PhD Scientific Days 2022

Pathology and Oncology III. (Poster discussion will take place in the Aula during the Coffee Break)

Dissection of spatial heterogeneity in relapsed/refractory follicular lymphoma using next-generation sequencing

Text of the abstract

Introduction: Follicular lymphoma (FL) is the most prevalent indolent non-Hodgkin lymphoma, characterized by a remitting-relapsing disease course, yet we have scarce information regarding the role of spatial heterogeneity during relapse. In this field, the analysis of circulating cell-free DNA (cfDNA) opened a new space enabling the genetic characterization of the total tumour burden of a patient.
Aims: In this feasibility study, we performed mutational profiling of a series of multiregional FL samples and liquid-biopsies to develop a next-generation sequencing pipeline for the investigation of tumour DNA and cfDNA samples.
Methods: Twelve tumour DNA or cfDNA samples were investigated from two FL patients. Sequencing libraries were prepared using a custom Agilent XTHS (Agilent, USA) gene panel spanning 174 genes and were sequenced on a NovaSeq6000 instrument (Illumina, USA). Variants were annotated using Ensembl Variant Effect Predictor. Results were analysed using R (v. 4.0.4).
Results: The first patient harboured mutations in the EP300, CARD11, CCND3, EGR2, SOCS1 and VMA21 genes at diagnosis. At progression, both compartments harboured a shared POU2F2 mutation and additional mutations exclusive to each region pointing to divergent evolution. In the pre-treatment cfDNA all mutations were detected characterizing both clones. The second patient harboured an EP300 and HLA-A mutated clone at diagnosis, which gained additional HIST1H4E, FOXO1 and EZH2 mutations at progression. During high-grade transformation, the tumour acquired additional SOCS1, TP53, MYC, TNFRSF14, RHOA and BCL7A mutations. In the liquid-biopsy sample obtained 6 months prior to transformation all mutations were identified. During second line treatment, all mutations were eliminated from cfDNA, but as the patient became refractory all previously identified mutations re-emerged in cfDNA.
Conclusion: We successfully designed an NGS pipeline for the comprehensive mutation profiling of FL, which we aim to use in forthcoming studies. Based on our preliminary results cfDNA based mutation profiling can dissect spatial heterogeneity and clonal dynamics during the disease course.
Funding: Semmelweis 250+ Excellence Scholarship (EFOP-3.6.3-VEKOP-16-2017-00009), EU Horizon2020 (No. 739593), National Research, Development and Innovation Office (K21_137948, TKP2021-EGA-24, TKP2021-NVA-15), Elixir Hungary