Using Targeted RNA Next Generation Sequencing and Differential Expression to Compare Primary Central Nervous System Lymphoma with CD+ DLBCL, Nodal DLBCL and EBV+ DLBCL
Authors: Abdulhaq Haifaa , Albitar Maher 
Published: November 2020

Introduction:Primary central nervous system lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL) with a tropism for the CNS microenvironment. Limited studies suggested differential expression of multiple extracellular matrix (ECM) and adhesions-related genes in PCNSL as compared to nodal DLBCL. We used next generation sequencing to evaluate expression profiles of 1408 genes and and performed differential expression profiling to compare PCNSL with nodal DLBCL, CD5+ DLBCL and EBV+ DLBCL. CD5+ DLBCL was selected because it is frequently ABC subtype. We also selected EBV+ DLBCL due to its poor outcome, similar to PCNSL.
Methods:RNA was extracted from 30 formalin-fixed paraffin-embedded (FFPE) tissue samples from PCNSL patients and 23 FFPE tissue samples from cases with lymph node DLBCL-NOS, 8 samples CD5+DLBCL, 14 samples EBV+ DLBCL.. We sequenced the RNA using a 1408 gene panel (Illumina). Next Generation Sequencing was based on hybrid capture methodology. Total number of reads per sample must exceed 5 million to be accepted. RNA levels were quantified using FPKM. Levels of expression of each of the 1408 genes were compared between groups. The T-score is used to show differences. Considering the number of analyzed samples and to correct for multiple testing, only T-score less or greater than 3 was considered clinically significant.
Results:PCNSL showed significant overexpression in 25 genes as compared with CD5+ DLBCL, while only 5 genes were highly expressed in PCNSL as compared with nodal DLBCL and 8 when compared with EBV+ DLBCL (Table below). Four genes were significantly less expressed in PCNSL as compared with CD5+ DLBCL, and only one gene when compared to nodal and EBV+ DLBCL. There was no significant difference in RNA expression of MYD88, CD79B, CARD11, KMT2D or SPP1 between PCNSL and DLBC-NOS, CD5+ DLBCL and EBV+ DLBCL . There was no significant difference between PCNSL and the other sub types in MYC, Ki67, BCL2, CD44, or CD274 (PD-L1) expression. Several genes emerged as highly expressed in PCNSL as compared with other subtypes of DLBCL (negative T-score). However, CLTC (Clathrin Heavy Chain) gene is uniquely highly overexpressed in PCNSL and not in any of the other 3 types of lymphoma. The CLTC gene is involved in anaplastic lymphoma as a partner gene for ALK (CLTC-ALK) in t(2,17) translocation. The SEPT2 (SEPTIN2) gene is uniquely highly expressed in PCNSL as compared with nodal and EBV DLBC lymphoma. SEPT2 gene plays significant role in Hodgkin lymphoma and in maintaining Reed-Stenberg cells . In contrast, VEGFC was significantly lower in PCNSL compared with nodal DLBCL.
Conclusions:RNA molecular profiling suggests significant difference between PCNSL and CD5+ DLBCL and less difference when compared with nodal and EBV+ DLBCL. The strikingly high expression of CLTC gene in PCNSL suggests possible targeting of this gene might present a specific therapeutic approach for PCNSL.
Figure

Disclosures
Abdulhaq: Novartis:Consultancy, Honoraria, Speakers Bureau;Astellas:Honoraria, Speakers Bureau;Amgen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Morphosys:Membership on an entity's Board of Directors or advisory committees, Research Funding;Oncopeptide:Research Funding.