Exploring the cell-free total RNA transcriptome in diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma patients as biomarker source in blood plasma liquid biopsies

Philippe Decruyenaere; Edoardo Giuili; Kimberly Verniers; Jasper Anckaert; Katrien De Grove; Malaïka Van der Linden; Dries Deeren; Jo Van Dorpe; Fritz Offner; Jo Vandesompele

doi:10.3389/fonc.2023.1221471

Exploring the cell-free total RNA transcriptome in diffuse large B-cell lymphoma and primary mediastinal B-cell lymphoma patients as biomarker source in blood plasma liquid biopsies

Front Oncol. 2023 Oct 25:13:1221471. doi: 10.3389/fonc.2023.1221471. eCollection 2023.

Authors

Philippe Decruyenaere^{1

2

3}, Edoardo Giuili^{4

5}, Kimberly Verniers^{2

3}, Jasper Anckaert^{2

3}, Katrien De Grove¹, Malaïka Van der Linden⁶, Dries Deeren⁷, Jo Van Dorpe⁶, Fritz Offner¹, Jo Vandesompele^{2

3}

Affiliations

¹ Department of Hematology, Ghent University Hospital, Ghent, Belgium.
² OncoRNALab, Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium.
³ Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.
⁴ Interuniversity Institute of Bioinformatics in Brussels (IB2), Free University of Brussels, Brussels, Belgium.
⁵ Department of Biotechnology and Pharmacy, University of Bologna, Bologna, Italy.
⁶ Department of Pathology, Ghent University Hospital, Ghent, Belgium.
⁷ Department of Hematology, Algemeen Ziekenhuis (AZ) Delta Roeselare-Menen, Roeselare, Belgium.

Abstract

Introduction: Diffuse large B-cell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) are aggressive histological subtypes of non-Hodgkin's lymphoma. Improved understanding of the underlying molecular pathogenesis has led to new classification and risk stratification tools, including the development of cell-free biomarkers through liquid biopsies. The goal of this study was to investigate cell-free RNA (cfRNA) biomarkers in DLBCL and PMBCL patients.

Materials and methods: Blood plasma samples (n=168) and matched diagnostic formalin-fixed paraffin-embedded (FFPE) tissue samples (n=69) of DLBCL patients, PMBCL patients and healthy controls were collected between 2016-2021. Plasma samples were collected at diagnosis, at interim evaluation, after treatment, and in case of refractory or relapsed disease. RNA was extracted from 200 µl plasma using the miRNeasy serum/plasma kit and from FFPE tissue using the miRNeasy FFPE kit. RNA was subsequently sequenced on a NovaSeq 6000 instrument using the SMARTer Stranded Total RNA-seq pico v3 library preparation kit.

Results: Higher cfRNA concentrations were demonstrated in lymphoma patients compared to healthy controls. A large number of differentially abundant genes were identified between the cell-free transcriptomes of DLBCL patients, PMBCL patients, and healthy controls. Overlap analyses with matched FFPE samples showed that blood plasma has a unique transcriptomic profile that significantly differs from that of the tumor tissue. As a good concordance between tissue-derived gene expression and the immunohistochemistry Hans algorithm for cell-of-origin (COO) classification was demonstrated in the FFPE samples, but not in the plasma samples, a 64-gene cfRNA classifier was developed that can accurately determine COO in plasma. High plasma levels of a 9-gene signature (BECN1, PRKCB, COPA, TSC22D3, MAP2K3, UQCRHL, PTMAP4, EHD1, NAP1L1 pseudogene) and a 5-gene signature (FTH1P7, PTMAP4, ATF4, FTH1P8, ARMC7) were significantly associated with inferior progression-free and overall survival in DLBCL patients, respectively, independent of the NCCN-IPI score.

Conclusion: Total RNA sequencing of blood plasma samples allows the analysis of the cell-free transcriptome in DLBCL and PMBCL patients and demonstrates its unexplored potential in identifying diagnostic, cell-of-origin, and prognostic cfRNA biomarkers.

Keywords: DLBCL; PMBCL; biomarkers; blood plasma; cell-free RNA; diffuse large B-cell lymphoma; liquid biopsy; primary mediastinal B-cell lymphoma.

Grants and funding

PD is funded by the Fund for Scientific Research Flanders (FWO, Belgium; 11H7523N). This work is supported by Ghent University (BOF-GOA REINFORCE). This study was also supported by the Fund for Scientific Research Flanders (FWO, Belgium; G0B2820N).